sun-lab.med.nyu.edusun-lab.med.nyu.edu/files/sun-lab/attachments/CPMB.Ch.01.E coli.pdf · cos site of action of phage lambda ter function. Cos site is cut by ter to yield two cohesive

CHAPTER 1Escherichia coli, Plasmids, and Bacteriophages

INTRODUCTION

Mastery of current DNA technology requires familiarity with a small number of basicconcepts and techniques. The goal of this chapter is to present this information

concisely, yet in enough detail to be useful when a procedure goes wrong. Section I isdevoted to Escherichia coli. Recipes are provided for media that support E. coli growth,as well as instructions for making the simple tools needed to work with bacterial cells.Growth of E. coli in liquid and solid media is then detailed. The final unit in Section Idescribes a few detailed aspects of E. coli biology learned from classical bacterial geneticstudies, the understanding of which is especially relevant to the techniques used in modernDNA work.

The remainder of the chapter discusses vectors used to introduce foreign DNA into E.coli. For the purposes of this chapter, vectors are said to be derived from plasmids, frombacteriophage lambda and related phages, or from filamentous phages. (Many modernvectors incorporate elements from more than one of these classes, and it is likely that thisclassification scheme will be hopelessly outdated by the time this chapter is revised.)Section II is concerned with plasmid vectors. Following a brief introduction to plasmidbiology, procedures are described for purifying small and large amounts of plasmid DNA(“minipreps” and large preps). Finally, procedures for reintroducing plasmid DNA intobacterial cells are described. Section III covers vectors derived from bacteriophages. Thebiology of bacteriophage lambda is first introduced, followed by detailed aspects ofbiology that are especially significant when lambda derivatives are used as cloningvectors. Protocols in this section describe techniques for manipulating lambda-derivedvectors, making single plaques, making and titering phage stocks, and isolating phageDNA. Finally, Section IV covers the biology and manipulation of vectors derived fromfilamentous phages.

This chapter will be meaningful primarily to readers with some knowledge of theprinciples of molecular biology. Several books on molecular biology are recommendedin the preface. For further advanced reading in the topics of this chapter, we recommendfive books, all from Cold Spring Harbor Laboratory: Methods in Molecular Genetics(Miller, 1972), Advanced Bacterial Genetics (Davis et al., 1980), The BacteriophageLambda (Hershey, 1971), Lambda II (Hendrix et al., 1983), and Experiments with GeneFusions (Silhavy et al., 1984).

Many terms and jargon used by molecular biologists are introduced in this chapter. Theseterms are italicized at their first mention, and are defined below.

alpha fragment peptide containing the aminoterminus of β-galactosidase, the lacZ geneproduct. Alpha fragments lack enzymatic ac-tivity, but can associate with omega fragments(see below) to form proteins whose β-galacto-sidase activity has been restored.alpha-complementation restoration of β-galac-tosidase activity to omega fragments by associa-tion with alpha fragments.

amplification increase in copy number ofsome plasmids that occurs when host proteinsynthesis is inhibited.cloning site site on a vector into which foreignDNA is inserted.competent state in which bacterial or yeastcells are able to take up foreign DNA (forexample, as the result of calcium treatment).

Supplement 59

Current Protocols in Molecular Biology (2002) 1.0.1-1.0.3Copyright © 2002 by John Wiley & Sons, Inc.

1.0.1

Escherichia coli,Plasmids, andBacteriophages

cos site of action of phage lambda ter function.Cos site is cut by ter to yield two cohesive ends(cos ends).dilution, 10x-fold a solution or suspension thatcontains 1⁄10

x as much (10−x as much) of thedissolved or suspended species as does thestarting liquid. For example, to “do a 102-folddilution” is to dilute a solution 100×.early-log phase period during the growth of aculture after the lag period. During early logphase growth, cells have begun exponentialgrowth.efficiency of plating (EOP) titer of bacterialcolonies or phage plaques under some experi-mental conditions divided by the titer of bacte-ria or phage obtained by growth on some ref-erence medium.exponential growth period during which thenumber of cells in the culture increases as anexponential function of time, that is, duringwhich cell number = ket.F factor genetic element found in some strainsof E. coli and related species. F encodes pro-teins used in formation of sex pili which allowits transfer from bacterium to bacterium.female strain strain that does not contain theF factor and that receives genetic informationwhen crossed with a strain containing F.helper phages bacteriophages that encode es-sential proteins and that allow other phageswhich do not encode these essential proteins togrow.incompatible phenomenon in which two plas-mids cannot replicate in the same cell withoutcontinual selection for both of them.incompatibility group consists of plasmidsthat cannot be maintained together in the samecell. Compatible plasmids belong to differentincompatibility groups.induction the onset of transcription of a newgene or operon, usually in response to someenvironmental stimulus. Phage induction orlysogenic induction describe the process inwhich prophage excise from the chromosomeof bacteria that harbor them and begin to growlytically.inoculation introduction of cells into a con-tainer of sterile growth medium.lag period period just after inoculation of aculture when cells have not yet begun to growexponentially.late-log phase last period of exponentialgrowth of a culture, after which growth slowsand then stops altogether due to nutrient ex-haustion or accumulation of waste products.lawn uniform layer of bacteria that covers thesurface of a plate.

log phase period during growth of a culture inwhich cells are growing exponentially.low-copy-number plasmids plasmids found inless than about 20 copies per cell when cellscontaining them are grown in rich medium.lysogen E. coli cell or strain that harbors adormant bacteriophage.male strain strain of bacteria that contains theF factor.male-specific phages bacteriophages thatonly grow on male strains because they adsorbto sex pili.marker detectable genetic difference betweenone organism and another (usually wild-type)organism of the same species.minimal medium growth medium for cellsthat contains only salts, vitamins, trace ele-ments, and simple compounds which serve ascarbon, nitrogen, and phosphorous sources.miniprep small-scale preparation or purifica-tion of some desired species, usually of plasmidor phage DNA.mobilization transfer of DNA from one cell toanother caused by a mobile genetic elementsuch as the F factor.multiplicity of infection (MOI) ratio of infect-ing bacteriophage to host cells.nonsense suppression the insertion of aminoacids into proteins at positions where transla-tion would normally not occur because themRNA contains a UAG (amber), UAA (ochre)or UGA nonsense codon.nonsense suppressor tRNA that inserts aminoacids at nonsense codons. The term is some-times used for the genes encoding these tRNAs.omega fragment protein containing the car-boxy-terminal fragment of β-galactosidase.This protein lacks enzymatic activity, but β-galactosidase activity can be restored when thepeptide is complexed with an alpha fragment.ori (origin) site on genome at which DNAreplication begins.outgrowth the growth of freshly transformedcells under nonselective conditions for enoughtime to allow proteins encoded by the foreignDNA to be expressed.overnight a small, freshly saturated liquid cul-ture of bacteria.packaging extract extract from special strainsof E. coli that contains bacteriophage lambdahead proteins, tail proteins, and packaging pro-teins. Phage DNA added to such an extract isassembled into phage particles.par site on some plasmids which ensures thateach daughter cell receives a plasmid copy.pilot protein protein in the coat of filamentousphages that helps phage DNA enter the cell.

Supplement 59 Current Protocols in Molecular Biology

1.0.2

Introduction

plates petri dishes filled with solid medium,used to grow separated bacterial colonies orplaques. The term is sometimes used to refer to96-well microtiter dishes.plating out the placement of bacteria or phageon plates so that colonies or plaques are formed.polylinker stretch of DNA that contains con-tiguous restriction sites.prophage dormant bacteriophage, usually in-tegrated into the host chromosome, that repli-cates with the host bacterium.relaxed control applies to plasmids whosereplication does not depend on the bacterial cellcycle.replicative form double-stranded circular fila-mentous phage DNA found inside infectedcells.replicator stretch of DNA on a phage or plas-mid that enables the phage or plasmid to repli-cate.rich medium growth medium that containscomplex organic molecules (peptides, nucleo-tides, etc.). Typical components of rich mediainclude tryptone (made from beef) and yeastextract (made from yeast).rolling-circle replication mechanism of repli-cation sometimes used by circular molecules inwhich DNA polymerase continually circum-navigates the template, and thus synthesizes along tail.satellite colonies small colonies that growaround a large colony on a plate containingselective medium. These are usually composedof cells unable to grow on selective medium,but which are able to grow near the large colonybecause the cells in the large colony neutralizethe selective agent.saturated culture culture of cells in liquid me-dium that has stopped growing because nutri-

ents are exhausted or because waste productshave accumulated.SOS response response of E. coli to DNAdamage or other treatments that inhibit DNAreplication. Lambda-derived phages are in-duced during this response.stringent control applies to plasmids whosereplication is synchronized with the E. coli cellcycle.temperate describes bacteriophages capableof lysogenic growth.transfection introduction of bacteriophageDNA into competent E. coli cells. Also de-scribes the introduction of any DNA (includingplasmid DNA) into cells of higher eukaryotes.transformation introduction of plasmid DNAinto E. coli or yeast. Also used to denote any ofa number of changes in cultured higher eu-karyotic cells to characteristics more typical ofcancer cells (immortal growth, loss of contactinhibition, etc.).

LITERATURE CITEDDavis, R., Botstein, D., and Roth, J.R. 1980. Ad-

vanced Bacterial Genetics. Cold Spring HarborLaboratory, Cold Spring Harbor, NY.

Hendrix, R., Roberts, J., Stahl, F., and Weisberg, R.1983. Lambda II. Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY.

Hershey, A.D. 1971. The Bacteriophage Lambda.Cold Spring Harbor Laboratory, Cold SpringHarbor, NY.

Miller, J. 1972. Methods in Molecular Genetics.Cold Spring Harbor Laboratory, Cold SpringHarbor, NY.

Silhavy, T., Berman, M.L., and Enquist, L.W. 1984.Experiments with Gene Fusions. Cold SpringHarbor Laboratory, Cold Spring Harbor, NY.

Current Protocols in Molecular Biology Supplement 59

1.0.3


SECTION IESCHERICHIA COLIEscherichia coli is a rod-shaped bacterium with a circular chromosome about 3 millionbase pairs (bp) long. It can grow rapidly on minimal medium that contains a carboncompound such as glucose (which serves both as a carbon source and an energy source)and salts which supply nitrogen, phosphorus, and trace metals. E. coli grows more rapidly,however, on a rich medium that provides the cells with amino acids, nucleotide precursors,vitamins, and other metabolites that the cell would otherwise have to synthesize. Thepurpose of this first section is to provide basic information necessary to grow E. coli. Amore detailed introduction to certain aspects of E. coli biology may be found in UNIT 1.4.

When E. coli is grown in liquid culture, a small number of cells are first inoculated intoa container of sterile medium. After a period of time, called the lag period, the bacteriabegin to divide. In rich medium a culture of a typical strain will double in number every20 or 30 min. This phase of exponential growth of the cells in the culture is called logphase (sometimes subdivided into early-log, middle-log, and late-log phases). Eventuallythe cell density increases to a point at which nutrients or oxygen become depleted fromthe medium, or at which waste products (such as acids) from the cells have built up to aconcentration that inhibits rapid growth. At this point, which, under normal laboratoryconditions, occurs when the culture reaches a density of 1–2 × 109 cells/ml, the cells stopdividing rapidly. This phase is called saturation and a culture that has just reached thisdensity is said to be freshly saturated.

With very few exceptions, bacterial strains used in recombinant DNA work are derivativesof E. coli strain K-12. Most advances in molecular biology until the end of the 1960scame from studies of this organism and of bacteriophages and plasmids that use it as ahost. Much of the cloning technology in current use exploits facts learned during thisperiod.

UNIT 1.1Media Preparation and Bacteriological Tools

Recipes are provided below for minimal liquid media, rich liquid media, solid media, topagar, and stab agar. Tryptone, yeast extract, agar (Bacto-agar), nutrient broth, andCasamino Acids are from Difco. NZ Amine A is from Hunko Sheffield (Kraft).

MINIMAL MEDIA

Ingredients for these media should be added to water in a 2-liter flask and heated withstirring until dissolved. The medium should then be poured into separate bottles withloosened caps and autoclaved at 15 lb/in2 for 15 min. Do not add nutritional supplementsor antibiotics to any medium until it has cooled to <50°C. After the bottles cool to below40°C, the caps can be tightened and the concentrated medium stored indefinitely at roomtemperature. All recipes are on a per liter basis.

M9 medium, 5×30 g Na2HPO4

15 g KH2PO4

5 g NH4Cl2.5 g NaCl15 mg CaCl2 (optional)

Supplement 59

Contributed by Karen Elbing and Roger BrentCurrent Protocols in Molecular Biology (2002) 1.1.1-1.1.7Copyright © 2002 by John Wiley & Sons, Inc.

1.1.1


M63 medium, 5×10 g (NH4)2SO4

68 g KH2PO4

2.5 mg FeSO4⋅7H2OAdjust to pH 7 with KOH

A medium, 5×5 g (NH4)2SO4

22.5 g KH2PO4

52.5 g K2HPO4

2.5 g sodium citrate⋅2H2O

Before they are used, concentrated media should be diluted to 1× with sterile water andthe following sterile solutions, per liter:

1 ml 1 M MgSO4⋅7H2O10 ml 20% carbon source (sugar or glycerol)and, if required:0.1 ml 0.5% vitamin B1 (thiamine)5 ml 20% Casamino Acids or

L amino acids to 40 µg/ml orDL amino acids to 80 µg/ml

Antibiotic (see Table 1.4.1)

RICH MEDIA

Unless otherwise specified, rich media should be autoclaved for 25 min. Antibiotics andnutritional supplements should be added only after the solution has cooled to 50°C orbelow. A flask containing liquid at 50°C feels hot but can be held continuously in one’sbare hands. All recipes are on a per liter basis.

H medium10 g tryptone8 g NaCl

Lambda broth10 g tryptone2.5 g NaCl

LB medium10 g tryptone5 g yeast extract5 g NaCl1 ml 1 N NaOH

The original recipe for LB medium (sometimes referred to as Luria or Lenox broth), doesnot contain NaOH. There are many different recipes for LB that differ only in the amount ofNaOH added. We use this formula in our own work. Even though the pH is adjusted to near7 with NaOH, the medium is not very highly buffered, and the pH of a culture growing in itdrops as it nears saturation.

NZC broth10 g NZ Amine A5 g NaCl2 g MgCl2⋅6H2OAutoclave 30 min5 ml 20% Casamino Acids


1.1.2

MediaPreparation and

BacteriologicalTools

Superbroth32 g tryptone20 g yeast extract5 g NaCl5 ml 1 N NaOH

TB (terrific broth)12 g Bacto tryptone24 g Bacto yeast extract4 ml glycerolAdd H2O to 900 ml and autoclave, then add to above sterile solution 100 ml of asterile solution of 0.17 M KH2PO4 and 0.72 M K2HPO4.

Tryptone broth10 g tryptone5 g NaCl

2× TY medium16 g tryptone10 g yeast extract5 g NaCl

TYGPN medium20 g tryptone10 g yeast extract10 ml 80% glycerol5 g Na2HPO4

10 g KNO3

SOLID MEDIA

Liquid media can be solidified with agar. For minimal plates, dissolve the agar in waterand autoclave separately from the minimal medium; autoclaving the two together willgive rise to an insoluble precipitate. For rich plates, autoclave the agar together with theother ingredients of the medium. Cool the agar to about 50°C and add other ingredientsif necessary. At this temperature, the medium will stay liquid indefinitely, but it willrapidly solidify if its temperature falls much below 45°C. Finally, pour the medium intosterile disposable petri dishes (plates) and allow to solidify.

Freshly poured plates are wet and unable to absorb liquid spread onto them. Moreover,plates that are even slightly wet tend to exude moisture underneath bacteria streaked onthem, which can cause the freshly streaked bacteria to float away. So for most applications,dry the plates by leaving them out at room temperature for 2 or 3 days, or by leaving themwith the lids off for 30 min in a 37°C incubator or in a laminar flow hood. Store dry platesat 4°C, wrapped in the original bags used to package the empty plates. Plates should beinverted when incubated or stored.

Minimal Plates

Autoclave 15 g agar in 800 ml water for 15 min. Add sterile concentrated minimal mediumand carbon source. After medium has cooled to about 50°C, add supplements andantibiotics. Pouring 32 to 40 ml medium into each plate, expect about 25 to 30 plates perliter.


1.1.3


Rich Plates

To ingredients listed below, add water to 1 liter and autoclave 25 min. Pour LB and Hplates with 32 to 40 ml medium, in order to get 25 to 30 plates per liter. Pour lambdaplates with about 45 ml medium for about 20 plates per liter.

H plates10 g tryptone8 g NaCl15 g agar

Lambda plates10 g tryptone2.5 g NaCl10 g agar

LB plates10 g tryptone5 g yeast extract5 g NaCl1 ml 1 N NaOH15 g agar or agarose

AdditivesAntibiotics (if required):Ampicillin to 50 µg/mlTetracycline to 12 µg/mlOther antibiotics, see Table 1.4.1

Galactosides (if required):Xgal to 20 µg/mlIPTG to 0.1 mMOther galactosides, see Table 1.4.2

TOP AGAR

Top agar is used to distribute phage or cells evenly in a thin layer over the surface of aplate. In a typical application, molten top agar is mixed with bacteria and the mixturepoured onto a plate to make a thin layer that is allowed to solidify. This layer of cells thengrows denser, forming the opaque lawn of cells. Top agar contains less agar than plates,and so stays molten for days when it is kept at 45° to 50°C. Top agarose is sometimesused when DNA is to be prepared directly from phage, and is also used when librariesare plated out to be screened by plaque lifting.

Prepare top agar in 1-liter batches, autoclave for 15 min to melt, cool to 50°C, swirl tomix, pour into separate 100-ml bottles, reautoclave, cool, and store at room temperature.Before use, melt the agar by heating in a water bath or microwave oven (see UNIT 1.11) thencool to and hold at 45° to 50°C.

H top agar10 g tryptone8 g NaCl7 g agar

LB top agar10 g tryptone5 g yeast extract5 g NaCl7 g agar


1.1.4



Lambda top agar10 g tryptone2.5 g NaCl7 g agar

Top agarose10 g tryptone8 g NaCl6 g agarose

STAB AGAR

Stab agar is used for storing bacterial strains (see UNIT 1.3). The recipe below is for 1 liter.

Stab agar10 g nutrient broth5 g NaCl6 g agar10 mg cysteine⋅Cl10 mg thymine

Thymine is included so that thy− bacteria can grow. Cysteine is thought to increase theamount of time bacteria can survive in stabs.

TOOLS

Inoculating Loops

Inoculating loops are used to move small numbers of bacteria or phage to a plate or to anew container of liquid medium. Inoculating loops may be purchased from any generalscientific supply company. However, most researchers prefer to use loops made in thelaboratory. These are made by inserting both ends of a 10-in. piece of 28-G platinum wireinto an inoculating loop holder (also widely available) and twirling the holder whiletugging on the middle of the wire with the point of a pencil (see Fig. 1.1.1).

Sterilize the loop by holding it in a bunsen burner flame until it is red hot. Cool the loopby touching it to a sterile portion of the surface of an agar plate until it stops sizzling.

Sterile Toothpicks

The broad side of flat wooden toothpicks may also be used for streaking out bacteria.Round wooden toothpicks, or the pointed end of flat toothpicks, are sometimes used topick individual colonies or phage plaques. To sterilize, place toothpicks in a small beaker,cover the beaker with foil, and autoclave. Alternatively, simply autoclave the whole boxof toothpicks and hold them in the middle when picking them up out of the opened box.It is convenient to put used toothpicks into another smaller beaker which, when full, iscovered with foil and autoclaved. Used toothpicks can be saved, reautoclaved, and usedagain (see Fig. 1.1.2).

Spreaders

Spreaders are used to distribute liquid containing bacterial cells evenly over a plate. Theyare made by heating and bending a piece of 4-mm glass tubing (see Fig. 1.1.3). Lessdurable spreaders can be made from a Pasteur pipet. Before each use, sterilize the spreaderby dipping the triangular part into a container of ethanol, passing the spreader through agas flame to ignite the ethanol, and letting the flame burn out. Be careful not to ignite the


1.1.5


ethanol in the container. Cool the spreader by touching it to the surface of an agar platethat has not yet been spread with cells.

Glass Beads

Although spreaders are useful for many applications, when processing large numbers ofplates it can become a time-consuming process. A popular practice is to use 4-mm glassbeads that have been sterilized. A half dozen or more beads distribute the liquid cultureon surface of the agar when the plate is shaken horizontally in all directions. The beadsare then discarded and the plate inverted and placed in the incubator. Stacks of plates canbe handled together when plating many culture samples.

Figure 1.1.1 Making a loop.

foil-coveredbeaker

steriletoothpicks

broad side down

Figure 1.1.2 Preparing sterile toothpicks.


1.1.6



Contributed by Karen ElbingClark & Elbing LLPBoston, Massachusetts

Roger BrentThe Molecular Sciences InstituteBerkeley, California

Figure 1.1.3 Making a spreader.


1.1.7


UNIT 1.2Growth in Liquid Media

BASICPROTOCOL 1

GROWING AN OVERNIGHT CULTURE

Small freshly saturated cultures of E. coli are called overnights. To make an overnight,remove the cap from a sterile 16- or 18-mm culture tube. Working quickly to minimizecontact of the tube with the possibly contaminated air, use a sterile pipet to transfer 5 mlof liquid medium into the tube. Inoculate the liquid with a single bacterial colony bytouching a sterile inoculating loop to the colony, making certain that some of the cellshave been transferred to the loop, and then dipping the loop into the liquid and shakingit a bit. Replace the tube’s cap, and place the tube on a roller drum at 60 rpm, 37°C. Growuntil the culture is freshly saturated (at a density of 1–2 × 109 cells/ml, which typicallytakes at least 6 hr).

BASICPROTOCOL 2

GROWING LARGER CULTURES

Larger cultures are generally inoculated with overnight cultures diluted 1:100. Use anErlenmeyer or baffle flask whose volume is at least 5 times the volume of the culture.Grow the culture at 37°C with vigorous agitation (∼300 rpm) to ensure proper aeration.If it is necessary to grow a culture without shaking (for example, if the strain istemperature-sensitive for growth and no low-temperature shaker is available), then, toensure that the cells get adequate aeration, grow the culture in an Erlenmeyer flask whosevolume is at least 20 times that of the culture.

BASICPROTOCOL 3

MONITORING GROWTH

With a Count Slide

Take a clean count slide (or hemacytometer) and cover it with a clean cover slip. Dip a0.1- or 1-ml pipet into the culture medium, allow a small drop of liquid to form on theend of the pipet, and touch it lightly to the surface of the slide at the periphery of the coverslip. The liquid will quickly spread under the cover slip. Put the slide on the stage of aphase-contrast microscope set to 400×, and focus on the cells. Each cell in a small squareis equivalent to 2 × 107 cells/ml (see Fig. 1.2.1).

Supplement 59


culture of E.coli

at 2.4 x 108 cells/mlviewed on a count slide

Figure 1.2.1 Monitoring growth via a hemacytometer or count slide.

1.2.1


With a Spectrophotometer

The concentration of cells in a culture can also be determined with a spectrophotometerby measuring the amount of 600-nm light scattered by the culture. The level of absorbance(A) at 600 nm will depend on the distance between the cuvette and the detector and willvary among spectrophotometers, often by a factor of 2. It is thus wise to calibrate eachinstrument by recording the OD600 (sometimes expressed as A600) of a culture that containsa known number of cells determined by some other method, such as observation on acount slide or titering for viable colonies (UNIT 1.3).

If the culture is visibly turbid, also measure a 10-fold dilution of it. For a culture grownin rich medium, a good rule of thumb is that each 0.1 OD unit is roughly equivalent to 1× 108 cells/ml.

Calculate the number of cells/ml from whichever suspension (the undiluted or the diluted)has an OD600 <1.




1.2.2

Growth in LiquidMedia

UNIT 1.3Growth on Solid Media

BASICPROTOCOL 1

TITERING AND ISOLATING BACTERIAL COLONIESBY SERIAL DILUTIONS

Bacteria are grown from single colonies to ensure that each cell in a population isdescended from a single founder cell, and thus to help ensure that each cell in the culturehas the same genetic makeup. One way to generate single colonies is to titer a culturewith serial dilutions and to pick colonies from one of the dilution plates. In this procedure,a small, measured amount of a bacterial culture is diluted into fresh liquid in another tube.A small amount of liquid is taken from this tube and diluted into another fresh tube. Thisprocess is repeated several times. Equal volumes of liquid are then taken from each of thedilution tubes and plated on petri plates. The plates are incubated overnight at 37°C;well-separated single colonies will arise on some of the dilution plates. The number ofliving bacteria in the culture is calculated from the number of colonies formed on thedilution plates.

A typical saturated culture contains 1 × 109 cells/ml. Phage suspensions can also be titered;a concentrated phage stock might typically contain 1 × 1011 phage/ml.

Materials

LB medium (UNIT 1.1)LB plates (UNIT 1.1)Sterile 16- or 18-mm-diameter culture tubes

1. Use pipets to introduce 5 ml LB medium into three sterile culture tubes. Line thetubes up, or label them so that they can be distinguished.

2. Using a pipettor, transfer 5 µl from the suspension of cells into the first tube of LBmedium. Set the vortexer to a mild setting and agitate the tube 5 sec.

3. Put a new tip on the pipettor and transfer 5 µl from the first tube of LB medium intothe second tube, and vortex the second tube. Take 5 µl from the second tube and repeatstep 3 until you have serial dilutions in all three tubes.

The first dilution tube now contains a 1 × 103-fold dilution, generated by diluting the cultureby a factor of one thousand (i.e., it contains 1 × 10−3 as many cells/ml as were present inthe original culture). The second tube contains a 1 × 106-fold dilution, generated by dilutingthe original culture by a factor of one million (i.e., it contains 1 × 10−6 as many cells/ml asthe original culture), etc.

Many investigators prefer to perform serial dilutions with different volumes and differentfactors of dilution. These parameters can be modified in steps 1 to 3.

4. Spread 100 µl of liquid from the culture and from each dilution tube onto separate,labeled, dry LB plates (as described on p. 1.3.2). Incubate overnight at 37°C.

During this incubation, each living bacterial cell will grow into a separate colony on theplate.

5. Count the colonies from these plates. Since only 100 µl was plated from the undilutedculture and from each dilution tube, each plate has 1⁄10 as many colonies on it as werepresent in each milliliter of liquid in the corresponding tube. Therefore, one candetermine the number of cells that were present per milliliter of the culture bycounting the number of colonies on a plate, and then multiplying that number by 10times the factor of dilution.

Supplement 59


1.3.1


For example, if 22 colonies were observed on the plate corresponding to the 1 × 106-folddilution, then the number of living cells in each milliliter of the original culture was 22 ×10 × 106, or 2.2 × 108 cells/ml.

6. Any of the single colonies may be saved for further use. Store plates at 4°C wrappedin parafilm or in the plastic sleeve in which the plates were supplied.

Commentary

Titering by serial dilutions is a good way to determine the number of any kind of livingorganism present in a suspension. The organisms do not even need to be able to grow intocolonies—i.e., the concentration of living bacteriophage in a tube can be determined bytitering with serial dilutions and counting the number of plaques made when an aliquotof each dilution is plated on a lawn of phage-sensitive bacteria (see UNIT 1.11).

It is sometimes useful to use smaller factors of dilution. Mixing 50 µl of the culture into5 ml medium will give dilutions of 100×. Mixing 100 µl into 900 µl will give dilutionsof 10×.

BASICPROTOCOL 2

ISOLATING SINGLE COLONIES BY STREAKING A PLATE

Another way to isolate single colonies is called streaking or streaking for single colonies.This method is easier and faster than serial dilutions for isolating single colonies, but itcannot be used to count the number of cells in a culture. An inoculum of bacteria is streakedacross one side of an agar plate with an inoculating loop or sterile toothpick. Theresterilized loop or a fresh toothpick is then passed once through the first streak andstreaked across a fresh part of the plate (see Fig. 1.3.1). This process is repeated at leastonce more, and the plate is incubated at 37°C until colonies become visible. If singlecolonies must be isolated from many bacteria, it is convenient to divide a plate into 4, 6,or 8 sectors and to streak for single colonies in each sector.

Figure 1.3.1 Streaking a plate.


1.3.2

Growth on SolidMedia

BASICPROTOCOL 3

ISOLATING SINGLE COLONIES BY SPREADING A PLATE

It is sometimes necessary to distribute a liquid culture of bacteria evenly over the surfaceof a plate (for example, when plasmid-containing colonies are to be isolated aftertreatment of cells with plasmid DNA and calcium chloride, UNIT 1.8). This is usually donewith a glass spreader. From 0.05 to 1 ml of liquid is pipetted onto a dry plate (see UNIT 1.1)and spread using a circular motion as shown in Figure 1.3.2. Alternatively, the edge ofthe spreader can be used to make a raster pattern on the plate’s surface. The plate can beturned at right angles and the process repeated. Evenly spread plates should be placed inthe incubator with the lids ajar until they are completely dry.

SUPPORTPROTOCOL 1

REPLICA PLATING

Replica plating is a convenient way to test many colonies for their ability to grow underdifferent conditions. In this technique, bacterial colonies are transferred from one plateto another in a way that maintains the original pattern of colonies. This technique hasmany applications to recombinant DNA work. As an example, consider the plasmidpBR322, which contains two antibiotic resistance genes, encoding resistance to ampicillinand tetracycline (see Fig. 1.5.9). A piece of foreign DNA cloned into the tetracyclineresistance gene inactivates it; cells that carry such a plasmid are ampicillin resistant buttetracycline sensitive. These cells can be identified by replica plating colonies fromampicillin-containing master plates onto plates containing tetracyline. Tetracyline-sensi-tive colonies can be identified by their inability to grow on the tetracycline plates, rescuedfrom the master plate, and analyzed further.

This procedure requires two specialty items: a replica block and sterile velvets. The replicablock is a wooden or metal cylinder that fits snugly inside a petri plate (see Fig. 1.3.3).One method for constructing these has been described by Adams (1965). A metal ring isused to secure the velvets to the block. Squares of velvet should be cut so as to cover thebase (a diameter of 14 cm is suggested). These velvets can be washed, autoclaved, andreused. If velvets are not available, pieces of sterile filter paper or disposable replica platescan be used (“Repli-Plate” Colony Transfer Pads, American Laboratories #59901).Replica plating also requires a master plate composed of well-separated colonies. Themaster plate can be a fresh plate onto which 50 to 100 colonies have been gridded (usingtoothpicks and the grid in Fig. 1.3.4), or it can be a plate on which bacteria were spreadthat have now grown up into well-separated colonies.

�

Figure 1.3.2 Spreading a plate.


1.3.3


Mark the top of the master plate to enable alignment with the grid. Press the plate downlightly onto the velvet. Do not bear down hard on the plate; pressing too hard will causethe colonies to run together on the velvet or may even cause the plate to collapse. Pressnew plates, oriented like the master plate, lightly onto the imprinted velvet to transfer thecolonies. As many as 10 plates per velvet can often be replica plated.

SUPPORTPROTOCOL 2

STRAIN STORAGE AND REVIVAL

Most strains of E. coli can be stored for years in stab vials, or indefinitely if frozen at−70°C. It is prudent to check the genetic markers of a strain revived from storage. Waysto verify the presence of other selective markers are described in Table 1.4.4.

1 2 3 4

6 7 8 9 105

12 13 14 15 1611

19 20 21 22 231817 24

27 28 29 30 312625 32

35 36 37 38 393433 40

42 43 44 45 4641

47 48 49 50

Figure 1.3.4 Grid for replica plating.

Figure 1.3.3 Replica block with velvet.


1.3.4


Stabs

Use airtight, autoclavable vials with rubber or Teflon caps (not cardboard). These areavailable from Wheaton Glassware and John’s Scientific (1⁄4-oz. Bijoux bottles, #15690-001). Fill the vials 2⁄3 full with stab agar (UNIT 1.1). Inoculate them with a single colony(see Fig. 1.3.5) by collecting most of the cells in the colony with an inoculating loop, thenrepeatedly poking the loop deeply into the agar. Leave the cap of the stab vial slightlyloose and incubate 8 to 12 hr at 37°C, or until cloudy tracks of bacterial growth are evident.Seal the vials tightly and store them in a cool (15° to 22°C), dark place. To revive a storedstrain, flame sterilize an inoculating loop (UNIT 1.1), allow it to cool, insert it into the stabagar, and move the loop around until a gobbet of bacteria-laden agar is stuck onto theloop. Smear the gobbet onto one section of an LB plate and streak for single colonies(Fig. 1.3.1).

Frozen Stocks

Add 2 ml of a mid-log culture or 1 ml of a freshly saturated culture to a stab vial or aNunc vial (Nunc #1087) containing 1 ml glycerol solution or DMSO solution (seerecipes). Vials can be stored at −20° to −70°C, but most strains remain viable longer ifstored at −70°C. Revive stored cells by scraping off splinters of solid ice with a toothpickor sterile pipet and streaking these splinters onto an LB plate (UNIT 1.1). Do not allow thecontents of the vial to thaw.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 4.

Glycerol solution65% glycerol (vol/vol)0.1 M MgSO4

0.025 M Tris⋅Cl, pH 8

Figure 1.3.5 Inoculating a stab vial.


1.3.5


DMSO solution7% dimethylsulfoxide (vol/vol)

The only advantage DMSO seems to have over glycerol for frozen stocks is that it is easierto pipet because it is less viscous. Use a bottle of reagent- or spectrophotometric-gradeDMSO that has been kept tightly sealed.

LITERATURE CITED

Adams, J.N. 1965. Automotive pistons for use as bases in velveteen replication. J. Bacteriol. 89:1627.

KEY REFERENCE

Lederberg, J. and Tatum, E.L. 1953. Novel genotypes in mixed cultures of biochemical mutants of bacteria.Cold Spring Harbor Symp. Quant. Biol. 18:75.

A primary reference in this field of research.




1.3.6


UNIT 1.4Selected Topics from Classical BacterialGenetics

Current cloning technology exploits manyfacts learned from classical bacterial genetics.This unit covers those that are critical to under-standing the techniques described in this book.

ANTIBIOTICSAntibiotics are chemicals that kill microor-

ganisms but are relatively nontoxic to eu-karyotic organisms. Antibiotics are very impor-tant for the techniques described in this book;genes encoding resistance to them are carriedon plasmid and phage vectors and cells thatcontain the vector are identified by their abilityto grow and form colonies in the presence ofthe antibiotic. Table 1.4.1 gives stock and work-ing concentrations, and mechanisms of actionof most of the antibiotics that are used in re-combinant DNA work.

Antibiotics are usually added to freshlyautoclaved solid medium after it has cooled tobelow 50°C. In an emergency, antibiotics canbe added directly to existing plates using thesame final concentration as above (assume thata plate 100 mm in diameter contains a totalmedium volume of 30 ml). Allow the antibiotictime to diffuse away from the very surface ofthe plate; an hour is usually sufficient. Sincemany antibiotics (especially ampicillin) losepotency at room temperature, plates are usuallystored at 4°C. In addition, rifampicin and tetra-cycline should be stored in the dark (see Table1.4.1).

THE LAC OPERONMany of the techniques described in this

book were made possible by early studies ofthe E. coli lac operon. The lac operon consistsof three genes—lacZ, lacY, and lacA (see Fig.1.4.1). When the cell grows on rich medium orglucose minimal medium, transcription isblocked by lac repressor (product of the neigh-boring lacI gene) which binds to a single site(operator) upstream of lacZ and prevents RNApolymerase from binding to the promoter.When the cell grows on medium that containslactose or certain related compounds (see Table1.4.2), lac repressor no longer binds the opera-tor, and RNA polymerase synthesizes a singlemRNA which encodes lacZ, lacY, and lacA. (Inthe wild-type lac operon, transcription initia-tion also requires the presence of a cAMP-CRPactivator complex; all lac promoters used in

cloning experiments are independent of thiscontrol.) Two of these genes are necessary forgrowth on lactose. lacY encodes a permeasewhich is necessary for the uptake of lactose andcertain related sugars. lacZ encodes a β-galac-tosidase, which cleaves lactose into glucose andgalactose, which the cell then utilizes. The thirdstructural gene, lacA, encodes an enzyme calledgalactoside acetyltransferase (GAT). This en-zyme is not required for lactose metabolism,but is thought to play a role in cellular detoxi-fication of nonmetabolizable lactopyranosides(Lewendon et al., 1995; Wang et al. 2002).

Alpha-ComplementationVectors such as the pUC series and the

M13mp series (see UNITS 1.5, 1.14, & 1.15) containa piece of DNA that encodes an alpha fragmentof β-galactosidase. These vectors exploit a phe-nomenon called alpha-complementation (seeFig. 1.4.2), which was discovered by Ullman,Jacob, and Monod in 1967. They showed thata cell that bears any of a number of deletionsof the 5′ end of the lacZ gene synthesizes aninactive C-terminal fragment of β-galactosi-dase, called an omega (ω) fragment. Similarly,a cell that bears a deletion of the 3′ end of lacZencodes an inactive N-terminal fragment ofβ-galactosidase called an alpha (α) fragment.However, if a cell contains two genes, onedirecting the synthesis of an alpha fragment,the other directing synthesis of an omega frag-ment, the β-galactosidase activity is observed.Many vectors incorporate a lac α-fragmentgene, which is small and easily manipulated.Exploitation of these vectors requires use of astrain carrying the complementing ω-fragmentgene to allow assembly of an active complex.This gene is often carried on an F′ plasmid (seebelow). When these vectors are used, cells con-taining them are grown on medium containingIPTG, which inactivates lac repressor and thusderepresses ω peptide synthesis, and Xgal,which is turned blue by the enzymatic activityof β-galactosidase (see Table 1.4.2). On thismedium, these vector-containing cells possessβ-galactosidase activity and turn blue.

Lactose AnalogsThere are many compounds related to lac-

tose that were first used for the biochemical andgenetic analysis of lac operon activity and are

Supplement 59

Contributed by Elisabeth A. Raleigh, Karen Elbing, and Roger BrentCurrent Protocols in Molecular Biology (2002) 1.4.1-1.4.14Copyright © 2002 by John Wiley & Sons, Inc.

1.4.1


Table 1.4.1 Antibiotics, Their Modes of Action, and Modes of Bacterial Resistancea

AntibioticbStockconc.

(mg/ml)

Finalconc.

(µg/ml)

Mode ofaction

Mode ofresistance

Ampicillinc 4 50 Bacteriocidal; only kills growing E.coli; inhibits cell wall synthesis byinhibiting formation of thepeptidoglycan cross-link

β-lactamase hydrolyzes ampicillinbefore it enters the cell

Chloramphenicol, in methanol

10 20 Bacteriostatic; inhibits proteinsynthesis by interacting with the 50Sribosomal subunit and inhibiting thepeptidyltransferase reaction

Chloramphenicol acetyltransferaseinactivates chloramphenicol

D-Cycloserine,d in 0.1 M sodium phosphate buffer, pH 8

10 200 Bacteriocidal; only kills growing E.coli; inhibits cell wall synthesis bypreventing formation of D-alaninefrom L-alanine and formation ofpeptide bonds involving D-alanine

Mutations destroy the D-alaninetransport system

Gentamycin 10 15 Bacteriocidal; inhibits proteinsynthesis by binding to the L6protein of the 50S ribosomal subunit

Aminoglycoside acetyltransferase andaminoglycosidenucleotidyltransferaseinactivate gentamycin; mutations inrplF (encodes the L6 protein) preventthe gentamycin from binding

Kanamycin 10 30 Bacteriocidal; inhibits proteinsynthesis; inhibits translocation andelicits miscoding

Aminoglycoside phosphotransferase,also known as neomycinphosphotransferase, aminoglycosideacetyltransferase, and aminoglycosidenucleotidyltransferase; inactivateskanamycin

Kasugamycin 10 1000 Bacteriocidal; inhibits proteinsynthesis by altering the methylationof the 16S RNA and thus an altered30S ribosomal subunit

Mutations prevent kasugamycin frombinding to the ribosome; mutationsdecrease uptake of kasugamycin

Nalidixic acid, pH to 11 withNaOH

5 15 Bacteriostatic; inhibits DNAsynthesis by inhibiting DNA gyrase

Mutations in the host DNA gyraseprevent nalidixic acid from binding

Rifampicin,e in methanol

34 150 Bacteriostatic; inhibits RNAsynthesis by binding to andinhibiting the β subunit of RNApolymerase; rifampicin sensitivity isdominant.

Mutation in the β subunit of RNApolymerase prevents rifampicin fromcomplexing; rifampicin resistance isrecessive

Spectinomycin 10 100 Bacteriostatic; inhibits translocationof peptidyl tRNA from the A site tothe P site

Mutations in rpsE (encodes the S5protein) prevent spectinomycin frombinding; spectinomycin sensitivity isdominant and resistance is recessive

Streptomycin 50 30 Bacteriocidal; inhibits proteinsynthesis by binding to the S12protein of the 30S ribosomal subunitand inhibiting proper translation;streptomycin sensitivity is dominant

Aminoglycoside phosphotransferaseinactivates streptomycin; mutations inrpsL (encodes the S12 protein)prevent streptomycin from binding;streptomycin resistance is recessive

Tetracycline,e in 70% ethanol

12 12 Bacteriostatic; inhibits proteinsynthesis by preventing binding ofaminoacyl tRNA to the ribosome A site

Active efflux of drug from cell

aData assembled from Foster (1983), Gottlieb and Shaw (1967), and Moazed and Noller (1987).bAll antibiotics should be stored at 4°C, except tetracycline, which should be stored at −20°C. All antibiotics should be dissolved in sterile distilled H2Ounless otherwise indicated. Antibiotics dissolved in methanol often can be dissolved in the less hazardous ethanol.cCarbenicillin, at the same concentration, can be used in place of ampicillin. Carbenicillin can be stored in 50% ethanol/50% water at −20°C.dD-cycloserine solutions are unstable. They should be made immediately before use.eLight-sensitive; store stock solutions and plates in the dark.


1.4.2

used in the cloning technology described in thisbook. These are described in Table 1.4.2.

THE F FACTORThe F (fertility) factor is a genetic unit

found in some strains of E. coli. Bacteria thatcontain the F factor are used in manytechniques described in this book, mainly fortwo reasons. First (as described later in thischapter), possession of F allows a cell to beinfected by vectors based on filamentousphages, which bind to cell surface structurescalled pili elaborated by F-containing cells.Second, defective lacZ genes that encode the ωfragment of β-galactosidase (described above)are commonly carried on F′ factors.

The F factor is found in three alternativeforms: as double-stranded, single-copy, circu-lar extrachromosomal plasmid DNA (F+); asplasmid DNA like F+ but also including otherbacterial genes (F′); and as a stretch of linear

DNA integrated into various sites on the bacte-rial chromosome (Hfr). Possession of the Ffactor confers on E. coli the ability to donateDNA in bacterial crosses (or matings). For thisreason cells that carry F are sometimes calledmale. F or F′ plasmids can transfer themselvesto other cells, and may occasionally causetransfer of other plasmids. This latter processis called mobilization. Mutations called tra pre-vent F from transferring itself or mobilizingother plasmids. Integrated Tra+ F factors (Hfr)can cause transfer of chromosomal DNA toother cells, but the recipient usually does notreceive the F sequence.

NONSENSE SUPPRESSORSSome vectors used in recombinant DNA

research (e.g., plasmid πVX and phage Charon4a) contain nonsense mutations in essentialgenes. These vectors must be propagated inspecial strains of E. coli. In these strains, trans-

z y a

z y a

z y a

z y a

the lac operonlac

repressor

terminationsite

RNApolymerase

z

z

ya

Figure 1.4.1 The first line shows the genes of the lac operon. lacZ, lacY, and lacA transcription isrepressed by lac repressor. In the next line, inactive repressor is no longer bound to the operator, andthe genes are being transcribed by RNA polymerase. In the third line, RNA polymerase transcribes theoperon. Finally, RNA polymerase encounters a transcription terminator and, in a reaction requiring rhoprotein, RNA polymerase is detached from the DNA and the nascent transcript.


1.4.3


lation of messages does not always stop whenthe ribosome encounters a chain terminationcodon (amber or ochre), but sometimes contin-ues, with a new amino acid installed at the endof the growing polypeptide. This process iscalled nonsense suppression and strains of E.coli in which it occurs are said to containnonsense suppressors. In a strain that containsan efficient or a “strong” suppressor, suppres-sion might occur 50% of the time an ambercodon is encountered.

The mechanism of nonsense suppression isa simple one: the cell contains a mutant speciesof tRNA in which the anticodon loop has mu-tated so that it base pairs with the UAG ambercodon or the UAA ochre codon. Nonsensesuppressors commonly used in cloning tech-nology are given in Table 1.4.3. UGA (opal)suppressors also exist but are rarely used.

GENETIC MARKERSGenetic markers in E. coli are named accord-

ing to the convention proposed by Demerec etal. (1966). All genes of a given strain are pre-sumed to be in the wild-type state unless oth-erwise noted in the genotype (see Table 1.4.4).Gene names have three italicized lowercaseletters, sometimes followed by an italic upper-case letter, and sometimes also followed by anitalic arabic number that specifies the precisemutation (allele) in question (e.g., lacY1, trp-31). The three-letter combination is usually amnemonic intended to suggest the function ofthe gene. Proper notation omits superscript +and − in a genotype, but these are sometimesused redundantly for clarity. Deletion muta-tions are described by ∆, followed by the namesof deleted genes in parentheses, followed by theallele number [e.g., ∆(lac-pro)X111]. The deltamay be replaced by “del” or “d.” Sometimes a

Table 1.4.2 Lactose Analogs Used in DNA Cloning Technology

Galactoside Stockconcentrationa Use Characteristics Reference

Isopropyl-1-thio-β-D- galactoside (IPTG)

100 mM Very effective inducer Nonmetabolizableinducer

Barkley andBourgeois,1978(pp. 177-220)

5-Bromo-4-chloro- 3-indolyl-β-D- galactoside (Xgal)

20 mg/ml(dissolved inN,N dimethylformamide)

Identification of lacZ+

bacteria, especially useful fordetecting β-galactosidasemade by recombinant vectors

Noninducing chromogenicsubstrate of β-galactosidase(cleavage of Xgal results inblue color); production ofblue color independent oflacY gene product

Miller, 1972

Orthonitrophenyl- β-D-galactoside (ONPG)

10 mM β-galactosidase assays Chromogenic substrate ofβ-galactosidase (cleavage ofONPG results in yellowcolor)

Miller, 1972(pp. 352-355)

6−Ο−β-D-Galacto- pyranosyl D-glucose (allolactose)

Inducer of the lactose operonin vivo; lactose is convertedinto allolactose byβ-galactosidase

Zabin andFowler, 1978(pp. 89-121)

Phenyl-β-D-galactoside (Pgal)

2 mg/ml Selection for lac constitutivemutants

Noninducing substrate ofβ-galactosidase; uptakepartly dependent on lacYgene product

Miller, 1978(pp. 31-88)

Orthonitrophenyl- β-D-thiogalacto- side (TONPG)

10 mM Selection for lac− mutants Transported into cells by lacpermease (the lacY geneproduct); inhibits cellgrowth at high concentration

Miller, 1978(pp. 31-88)

aStock solutions should be dissolved in sterile water unless otherwise noted.


1.4.4

Selected Topicsfrom Classical

Bacterial Genetics

ß-gal monomer ß-gal omega fragment

ß-gal alpha fragmentalpha-complementing ß-gal

ω α α

ω

pUCplasma

ω α

bacterial cell

αα

α

ωω

ω

F′ lacZ M15

Figure 1.4.2 Alpha-complementation.

Table 1.4.3 Commonly Used Nonsense Suppressorsa

Suppressor Mappositionb

Type ofsuppressor

Amino acidinserted

tRNAgene

supD (su1) 43 Amber Serine serUsupE (su2) 16 Amber Glutamine glnUsupF (su3) 27 Amber Tyrosine tyrTsupB (suB) 16 Ochre/amber Glutamine glnUsupC (suC) 27 Ochre/amber Tyrosine tyrTaData compiled from Bachmann (1983) and Celis and Smith (1979).bGiven in minutes; see Bachmann (1983) for description.


1.4.5


phenotype designation (see box) in parenthesesfollows the genotype designation, if the formeris not obvious from the latter [e.g., rpsL104(Strr)]. However, this usage is by no meansuniversal.

Table 1.4.5 lists commonly used geneticmarkers, with methods for verifying their pres-ence or absence in bacterial cells. Genotypes ofseveral strains used for different applicationsare listed in Table 1.4.6.

GENOTYPE AND PHENOTYPEGenotype indicates what genes are mutated

in a strain. A genotype is a theoretical constructdescribing a genetic constitution that wouldexplain the phenotype of the strain. It is derivedfrom considerations of the strain’s behavior andancestry.

Phenotype describes the observable behav-ior of the strain—e.g., Lac− fails to grow onlactose as a sole carbon source. Phenotypes arein Roman type, the first letter is capitalized, andthe letters are always followed by superscript+ or − (sometimes r, resistant, or s, sensitive).A phenotype is a datum to be explained.

Genotype and phenotype names are usuallyrelated, but the relationship is not always obvi-ous. Examples are provided in Table 1.4.4.

DNA RESTRICTION,MODIFICATION, ANDMETHYLATION

This section and the next two describe E.coli functions that can prevent cloning the se-quence of interest. E. coli has at least fourrestriction systems that identify foreign DNAand destroy it. These systems, encoded byhsdRMS, mcrA, mcrB, and mrr, can be avoidedby using host strains in which they are disabledby mutation. Restriction of DNA and the con-tent of methylated bases in the DNA are inter-related as described below. To select the appro-priate strain, it is necessary to know the contentof methylated bases in the DNA to be cloned.

The EcoK restriction system, encoded bythe hsdRMS genes, is the best understood ofthe E. coli restriction mechanisms (Bickle,1982). It attacks DNA that carries the site:

5′ A mA C N N N N N G T G C 3′3′ T T G N N N N N C mA C G 5′

Table 1.4.4 Examples of Gentotype and Phenotype Nomenclature

Genotype Phenotype Description of phenotype

Some straightforward examples

trp-31 Trp− Requires tryptophan for growth on minimal media

uvrA UVs Sensitive to UV light

recA Rec− Recombination defective

Some common examples that are not so straightforward

supE44 Sup+ Carries a tRNA suppressor gene. The mutant gene product, not thewild type, suppresses nonsense mutations; wild type is indicated assup0, Sup−, and does not suppress

rpsL104 Strr Resistant to streptomycin (this makes a mutant ribosomal protein,small subunit, the target of the drug)

rpsE Spcr Resistant to spectinomycin (also codes for a ribosomal protein, adifferent one)

gyrA Nalr Resistant to nalidixic acid (the mutation affects DNA gyrase)

One mutation may create several phenotypes

dam-3 Dam−,2-APs

UVs

DNA not methylated at adenines in GATCSensitive to 2-aminopurineSensitive to UV light

hsdS EcoK R−,EcoK M−

Neither restricts nor modifies DNA that enters the cell

Some mutations lead to counterintuitive phenotypes

recD ExoV− butRec+

Exonucleolytic activity of the RecBCD protein is defective, but therecombinational activity is intact


1.4.6


Bacterial Genetics

Table 1.4.5 Commonly Used Genetic Markers and How to Test Thema

Nutritional markers

Streak or replica plate colonies of the strain onto plates with and without thenutrient to be tested, but which contain all other necessary nutrients.

Antibiotic resistance markers

Streak or replica plate colonies of the strain onto plates with and without theantibiotic.

Other markers

lacZ+ Streak strain on an LB plate with Xgal and IPTG (UNIT 1.4). Colonies should turnblue. Colonies of control lacZ− strain should not turn blue.

lacZ∆M15b Transform strain with pUC plasmid and with control plasmid such as pBR322.Streak transformants onto LB/ampicillin plate with Xgal and IPTG. Coloniesbearing pUC plasmid should turn blue, while colonies bearing pBR322 shouldnot.

F+ or F′ Spot M13 phage onto a lawn of the cells. Small plaques should appear (see UNIT 1.15).

recA Using a toothpick, make a horizontal stripe of cells across an LB plate. Alsomake a stripe of recA+ control cells. Cover half of the plate with a piece ofcardboard, and irradiate the plate with 300 ergs/cm2 of 254 nm UV light from ahand-held UV source (typically 20 sec exposure from a lamp held 50 cm over theplate). recA− cells are very sensitive to killing by UV light, and recA− cells in theunshielded part of the plate should be killed by this level of irradiation.

recBCD Spot dilutions of λ gam−(UNIT 1.9) on a lawn of cells side by side with dilutionsof λ gam+. The gam− plaques should be almost as big as the gam+ plaques.

hsdS− (1) Use the strain and a wild-type strain to plate out serial dilutions of a λ-likephage stock grown on an hsdS− or hsdR− host. If the phage stock came from anhsdS− host, then it should make plaques with 1 × 104 to 1 × 106 higher efficiencyon the putative hsdS− host than on a wild-type host. If the plate stock came froman hsdR− (hsdS+ hsdM+) host, it should make plaques with the same efficiency onboth strains.

(2) Suspend one of the fresh plaques from the putative hsdS− host in 1 mllambda dilution buffer. Titer this suspension on the putative hsdS− strain and on awild-type strain. The suspension should make plaques at 1 × 104 to 1 × 106

higher efficiency on the hsdS− strain than on the wild-type strain. One plaquecontains ∼1 × 107 phage.

hsdR− (1) Perform step 1 described above, using a plate stock made from an hsdS− host.

(hsdS+

hsdM+)(2) Suspend one of the fresh plaques in 1 ml lambda dilution buffer. Titer thissuspension on the putative hsdR– strain and on a wild-type strain. Thissuspension should make plaques with the same efficiency on the hsdR− as on awild-type strain.

dam Transform the strain and a wild-type strain with a plasmid that containsrecognition sites for the enzymes MboI or BclI. Prepare plasmid DNA from bothstrains and verify that plasmid DNA isolated from the dam− strain is sensitive todigestion by the enzyme.

dcm Transform the strain and a wild-type strain with a plasmid that containsrecognition sites for ScrFI. Prepare plasmid DNA from both strains to verify thatonly plasmid DNA from the dcm strain is fully sensitive to digestion by theenzyme. Half of the ScrFI sites will be cut even when the DNA isdcm-methylated.

lon Streak LB plate for single colonies. Also streak a control plate of a wild-typestrain. Incubate at 37°C. Colonies of the lon– strain should be larger, glistening,and mucoidal.

aCommonly used protocols in this table are media preparation (UNIT 1.1), streaking and replicating a plate (UNIT 1.3), andgrowing lambda-derived vectors (UNIT 1.12).bEncodes omega fragment of β-galactosidase.


1.4.7


and results in double-strand cleavage at a vari-able distance from the site, leading eventuallyto degradation of the resulting fragments. DNAis not attacked if it lacks the site, or if the siteis present but methylated at the adenines shown(mA).

The EcoK enzyme is both genetically andenzymatically complex. The HsdR, HsdM, andHsdS subunits are required for restriction of anunmethylated substrate. The same complexwill methylate the same substrate, but at a veryslow rate, so that an unmethylated target rarely

survives. A substrate methylated on only onestrand (hemimethylated) will be methylated onthe other strand by the three-protein complex,but will not be cut. HsdM and HsdS togethercan methylate either an unmethylated or ahemimethylated substrate. The three-proteincomplex is inactive for restriction if any of thethree subunits is defective, but can still methy-late if HsdR is defective.

In summary, a strain defective in the hsdRgene is described as having the phenotypeHsdR−M+ (or, equivalently, EcoK R−M+ or

Table 1.4.6 Commonly Used Escherichia coli Strains

Straina Genotype Referenceb

AR58 sup0 galK2 galE::Tn10 (λcI857 ∆H1 bio− uvrB kil− cIII−) Strr A. Shatzman, pers comm.d

AR120 sup0 galK2 nad::Tn10 (Tetr) (λcI+ ind+ pL-lacZ fusion) Strr A. Shatzman, pers comm.d

AS1f endA1 thi-1 hsdR17(rK−mK

+) supE44 (λcI+) A. Shatzman, pers comm.d

BNN102f C600 hflA150 chr::Tn10 mcrA1 mcrB Young and Davis, 1983c

BW313g Hfr lysA− dut ung thi-1 recA spoT1 Kunkel et al., 1987c,d

C600 thi-1 thr-1 leuB6 lacY1 tonA21 supE44 mcrA Appleyard, 1954c,e

CJ236g dut1 ung1 thi-1 relA1/pCJ105 (Cmr) Kunkel et al., 1987c; Joyce andGrindley, 1984

DH1 recA1 endA1 thi-1 hsdR17 supE44 gyrA96 (Nalr) relA1 Hanahan, 1983c; D. Hanahan,pers. comm.d,e

DH5αF′h F′/endA1 hsdR17(rK−mK

+) supE44 thi-1 recA1 gyrA (Nalr) relA1 ∆(lacZYA-argF)U169 (m80lacZ∆M15)

See DH1 references

DK1 hsdR2 hsdM+ hsdS+ araD139 ∆(ara-leu)7697 ∆(lac)X74 galU galK rpsL (Strr) mcrA mcrB1 ∆(sr1-recA)306

D. Kurnit and B. Seed, pers.comm.d,e

ER1451 F′ traD36 proAB lacIq ∆(lacZ)M15/endA gyrA96 thi-1 hsdR2 (or hsdR17) supE44 ∆(lac-proAB) mcrB1 mcrA

Raleigh et al., 1988d,e

HB101i ∆(gpt-proA)62 leuB6 thi-1 lacY1 hsdSB20 recA rpsL20 (Strr) ara-14 galK2 xyl-5 mtl-1 supE44 mcrBB

Boyer and Roulland-Dussoix,1969c,d,e

JM101j F′ traD36 proA+ proB+ lacIq lacZ∆M15/supE thi ∆(lac-proAB) Yanisch-Perron et al., 1985c,d,e

JM105j F′ traD36 proA+ proB+ lacIq lacZ∆M15/∆(lac-pro)X111 thi rpsL (Strr) endA sbcB supE hsdR

See JM101 references

JM107j F′ traD36 proA+ proB+ lacIq lacZ∆M15/endA1 gyrA96 (Nalr) thi hsdR17 supE44 relA1 ∆(lac-proAB) mcrA


JM109j F′ traD36 proA+ proB+ lacIq lacZ∆M15/recA1 endA1 gyrA96 (Nalr) thi hsdR17 supE44 relA1 ∆(lac-proAB) mcrA


K38 HfrC (λ) Russel and Model, 1984

KM392 hsdR514(rK−mK

+) supE44 supF58 lacY galK2 galT22 metB1 trp55 mcrA ∆lacU169 proC::Tn5

T. St. John, pers. comm.d; K.Mooree

LE392 hsdR514(rK−mK

+) supE44 supF58 lacY galK2 galT22 metB1 trp55 mcrA

Borck, et al., 1976c; N. Murray,pers. comm.d; L. Enquiste

MC1061 hsdR2 hsdM+ hsdS+ araD139 ∆(ara-leu)7697 ∆(lac)X74 galE15 galK16 rpsL (Strr) mcrA mcrB1

Casadaban and Cohen, 1980c; M.Casadaband,e

MM294 endA thiA hsdR17 supE44 Backman et al., 1976c; M. Meselsond,e

continued


1.4.8


Bacterial Genetics

RK– MK

+ ; see box): it will methylate newly intro-duced DNA but will not restrict it. However, astrain defective in either hsdM or hsdS willneither restrict nor methylate, and has the phe-notype HsdR−M− (or EcoK R−M− or RK

– MK– .

In contrast with EcoK, the other three re-striction systems of E. coli K-12—mcrA, mcrB,and mrr—specifically attack DNA that is meth-ylated at particular sequences, rather than DNAthat is not. Either methylated cytosine residuesor methylated adenine residues can create prob-lems (see below).

The action of either mcrA or mcrB reducesthe number of clones recovered from librariesmade with genomic DNA from other organ-

isms, and leads to bias against recovery ofspecific fragments from those libraries(Raleigh et al., 1988; Whittaker et al., 1988;Woodcock et al., 1988, 1989; mrr has not beentested). For McrB there is evidence that a nu-clease is responsible for these effects (E. Suth-erland and E.A. Raleigh, unpub. observ.), butno such evidence is available for the other twosystems.

Even without biochemical characterization,something can be said of the recognition sitesfor these systems. McrA restricts DNA modi-fied by the HpaII (5′ CmCGG) methylase andpossibly other methylases. McrB restricts DNAmodified by any one of 14 other modification

NM539k supF hsdR (P2cox3) Frischauf et al., 1983c; Lindahland Sunshine, 1972d; N. Murraye

P2392 hsdR514(rK−mK

+) supE44 supF58 lacY galK2 galT22 metB1 trp55 mcrA (P2)

L. Klickstein, pers. comm.d

PR722 F′ ∆(lacIZ)E65 pro+/proC::Tn5 ∆(lacIZYA)U169 hsdS20 ara-14 galK2 rpsL20 (Strr) xyl-5 mtl-1 supE44 leu

P. Riggs, pers.comm.d

Q359 hsdR− hsdM+ supE tonA (ϕ80r) (P2) Karn et al., 1980c,d,e

RR1 ∆(gpt-proA)62 leuB6 thi-1 lacY1 hsdSB20 rpsL20 (Strr) ara-14 galK2 xyl-5 mtl-1 supE44 mcrBB

Bolivar et al., 1977

Y1088l supE supF metB trpR hsdR− hsdM+ tonA21 strA ∆lacU169

mcrA proC::Tn5/pMC9Huynh et al., 1985c; Miller et al.,1984d; R. Younge; M. Calos (pMC9)e

Y1089l ∆lacU169 proA+ ∆(lon) araD139 strA hflA150 chr::Tn10/pMC9 See Y1088 references

Y1090l ∆lacU169 proA+ ∆(lon) araD139 strA supF trpC22::Tn10 mcrA/pMC9

See Y1088 references

aThe original E. coli K-12 strain was an F+ λ lysogen, but most K-12 derivatives in common use have been cured of the F factor and prophage and theseare indicated only when present. All other genes in these strains are presumed to be wild-type except for the genotype markers noted in the secondcolumn.bReference for all mcr and mrr genotypes is Raleigh et al., 1988.cReference for genotype of strain.dSource of additional genotype information.eThought to be responsible for original strain construction.fAS1 is also known as MM294cI+. BNN102 is also known as C600 hflA.gBoth CJ236 and BW313 are commonly used in oligonucleotide-directed mutagenesis. pCJ105, the plasmid CJ236 carries, is not relevant for thisapplication.hThree strains are in circulation. DH5 is a derivative of DH1 that transforms at slightly higher efficiency. DH5α and DH5αF′ are derivatives that carrya deletion of the lac operon and a Φ80 prophage that directs synthesis of the omega fragment of β-galactosidase. DH5αF′ carries an F′ factor as well.DH5α and DH5αF′ are proprietary strains and the cells are prepared in some way that allows them to be transformed with slightly higher efficiency thanDH5.iIn this strain, the area of the chromosome that contains the hsd genes was derived from the related B strain of E. coli.jThe continued presence of the F′ factor in JM strains can be insured by starting cultures only from single colonies grown on minimal plates that do notcontain proline. These strains encode the omega fragment of lacZ and are frequently used with vectors that direct the synthesis of the lacZ alpha fragment.These strains are frequently used with M13 vectors for DNA sequencing (UNITS 1.14, 1.15, & 7.4).kIt is not known whether this strain has markers other than those listed.lpMC9, the plasmid in the Y strains listed here, directs the synthesis of large amounts of lac repressor. It also confers resistance to tetracycline andampicillin (Lebrowski et al., 1984, EMBO. J. 3:3117-3121).

Table 1.4.6 Commonly Used Escherichia coli Strains, continued

Straina Genotype Referenceb


1.4.9


methylases, which led to the suggestion that theMcrB recognition site is 5′ GmC (Raleigh andWilson, 1986). Mrr restricts DNA modified bythe HhaII (5′ GmANTC) or PstI (5′ CTGCmAG)methylases, but not that modified by the EcoRImethylase, among others (Heitman and Model,1987).

Many commonly used E. coli strains areMcrA−; including (from Table 1.4.5) BNN102(also known as C600hflA), C600, JM107,JM109, LE392, Y1088, and Y1090. Of thestrains listed in Table 1.4.5, only BNN102,HB101, and MC1061 are McrB−; and onlyHB101 is Mrr− (see also Raleigh et al., 1988).

A strain should be used which lacks theappropriate methyl-specific restriction sys-tem(s) when cloning genomic DNA from anorganism containing methylated bases. Allmammals and higher plants, and manyprokaryotes, contain methylcytosine (Ehrlichand Wang, 1981), so McrA−B− strains shouldbe used for libraries of DNA from these organ-isms. Bacteria and lower eukaryotes may con-tain methyladenine, so Mrr sensitivity shouldbe considered. However, the important experi-mental organisms Drosophila melanogasterand Saccharomyces cerevisiae contain no de-tectable methylated bases.

In addition, any time DNA is methylated invitro during a manipulation, an appropriaterestriction-deficient host should be used asDNA recipient. Methylases are used to generatenovel restriction enzyme specificities or to pro-tect cDNA from subsequent digestion (see UNITS

3.1 & 5.6). For example, the AluI methylase(M.AluI) is sometimes used to protect HindIIIsites. McrB will restrict DNA modified byM.AluI.

Once the DNA introduced into E. coli hasbeen replicated, the foreign methylation patternwill be lost (and the E. coli methylation patternwill be acquired) unless the clone carries amethylase activity. Once successfully intro-duced, clones can be freely transferred amongMcr+ Mrr+ E. coli strains, since the methylationpattern will no longer be foreign. It is importantthat the clone be passed through an HsdM+

strain before trying to introduce it into anHsdR+ strain.

The normal methylation pattern of E. coliDNA is the product of three methylases. TheEcoK methylase modifies the sequences indi-cated above. The dam and dcm gene productsare also methylases (Marinus, 1987). The rec-ognition sites for these are:

dam 5′ G mA T C 3′3′ C T mA G 5′

dcm 5′ C mC A G G 3′3′ G G T mC C 5′

dcm 5′ C mC T G G 3′3′ G G A mC C 5′

These modifications will render DNA resis-tant or partially resistant to some restrictionendonucleases used for in vitro work (see Table3.1.1), such as MboI and BclI (for Dam-modi-fied DNA) or EcoRII (for Dcm-modifiedDNA). The Dam and Dcm methylases are notassociated with any E. coli restriction function.Loss of Dam and/or Dcm methylation will notmake the DNA sensitive to EcoK restriction,although loss of K modification will. However,Dam and Dcm modification confer sensitivityto Mrr and Mcr analogues in Streptomycesspecies (MacNeil, 1988).

RECOMBINATION AND ITSEFFECTS ON CLONED DNAINSERTS

During propagation in E. coli, DNA insertedinto vectors is sometimes rearranged by theproteins involved in DNA recombination. For-tunately, although the genetics and enzymologyof recombination in E. coli are still not wellunderstood, there are mutant strains availablethat can provide solutions to two common clon-ing problems.

Problem 1. The DNA contains dispersedrepeated sequences. Recombination occurs be-tween these repeated sequences, causing lossof pieces of the DNA (see Fig. 1.4.3).

For plasmid libraries, this problem can besolved by propagating the DNA in a recA− host,where homologous recombination does not oc-cur. For libraries made using λ-derived vectors,the vector must also be recombination-defec-tive (red). However, only about 30% to 50% ofthe cells are viable in such a strain, and libraries,particularly phage libraries, may be hard topropagate. Phage λ vectors that are red will notmake high-titer lysates in recA strains, and redgam phage will not grow at all, unless therecBCD enzyme is also inactivated (see below).Many λ vectors are red gam to make use of theSpi− selection or to make room for larger insertpieces (see UNIT 1.10).

Problem 2. The inserted DNA containsclosely spaced inverted repeat sequences (pal-indromes or interrupted palindromes). Suchstretches of DNA are not stably propagated ineither phage or plasmid vectors. Available


1.4.10


Bacterial Genetics

knowledge is consistent with the idea that large(>300-bp) palindromes can sometimes form analternative, hairpin structure that resembles anintermediate found in normal recombinationcalled a Holliday junction, and are then actedupon by the host recombination system in sucha way that the hairpin is eliminated or madesmaller.

There are strains of bacteria from whichphage and plasmid clones containing palin-dromes are recovered at higher frequency.These bacteria have inactivated exonucleaseV (ExoV; encoded by the recB, recC, andrecD genes) or the SbcC product (encoded bythe sbcC gene). Many strains permissive forpalindromes have defects in recB recCcombined with a defect in sbcA (which prob-ably encodes the RecE protein) or sbcB (whichencodes exonuclease I).

Involvement of ExoV (RecBCD enzyme) inpalindrome stabilization was first noticed byLeach and Stahl (1983) using artificially con-structed palindromes in λ phages, and hosts

mutant in recB, recC, and sbcB. Because recBrecC strains are sick, they tend to accumulatetwo additional mutations, one in sbcB (the genefor exonuclease I; suppressor of recBC), andone in sbcC, the biochemical nature of whichis unknown (Lloyd and Buckman, 1985). To-gether these mutations restore recombinationand increase cell viability. Wertman et al.(1986) and Wyman et al. (1986) found that boththe recB recC defect and the sbcB defect inde-pendently contributed to stabilization of clonedpalindromes in λ libraries.

Involvement of recD was first investigatedby Wertman et al. (1986) and Wyman et al.(1986). recD codes for the nuclease activity ofExoV, as distinct from the recombination activ-ity of that enzyme. Strains mutant in recD aloneare Rec+ and healthy, and are not known toaccumulate secondary mutations. Such strainswere the best hosts for palindrome stabilizationusing λ-derived phages in the studies citedabove.

A B C D E F G H A B C

CBA

F E D

A B C

HG

F ED

G

H

A B C

BA C

Figure 1.4.3 Recombination between homologous direct repeats.


1.4.11


The effect of sbcC was examined by Chalkeret al. (1988), who found that sbcC alone wasbetter at maintaining palindromes in λ thanrecD alone and better than recB recC sbcB, butthat recD sbcC strains attained the highest pal-indrome stability (recA recD sbcC strains alsomaintained the palindrome). A further advan-tage of sbcC mutant strains is that palindromeswere maintained in plasmids as well as phage(Chalker et al., 1988), whereas ExoV− deficientmutants are poor hosts for plasmids regardlessof palindrome content (see below).

EFFECTS OFRECOMBINATION-DEFECTIVESTRAINS ON VECTORS

Lambda-derived vectors or clones that arered gam (see UNIT 1.10, especially the spi− selec-tion) must be propagated on ExoV− hosts, be-cause the long linear multimers that are thenormal substrate for lambda packaging are ex-onucleolytically degraded by ExoV in the ab-sence of the Gam protein (Stahl, 1986). Thesephage will grow well on ExoV− RecA+ hosts,reasonably well on ExoV− RecA− hosts, verypoorly on ExoV+ RecA+ (the phage are pack-aged by an alternative packaging mechanismusing circular dimers produced by recombina-tion that depends on E. coli proteins), and notat all on ExoV+ RecA−. With ExoV− RecA+

hosts, clones carrying a recombination hot spotcalled a Chi site (5′ GCTGGTGG 3′) will out-grow those that don’t, resulting in a biasedlibrary. The ExoV− RecA+ host can be recBCsbcB or recD, and the ExoV− RecA− host canbe recB recC sbcB recA or recD recA.

Most cloning plasmids, including ColE1 de-rivatives like pBR322, and p15A derivatives likethe pACYC vectors, are very unstable in ExoV-deficient strains unless selection is maintained.Moreover, they are often unstable or difficult tomaintain even with selection. Both recB recCsbcA strains (Basset and Kushner, 1984) andrecD strains (Biek and Cohen, 1986, and refer-ences therein) behave this way. However, RecA−

suppresses this effect. Instability is probably dueto recombination-initiated rolling-circle repli-cation of the plasmids that leads to synthesis oflong linear multimers, which fail to segregateproperly at cell division (Silberstein and Cohen,1987). The problem is particularly severe withvery high-copy-number ColE1 derivatives suchas pUC vectors, which may be impossible toestablish at all in recD strains (E.A. Raleigh,unpublished observation).

From the above considerations, a universalhost strain for phage and plasmid cloning vec-

tors would have the markers recA recD sbcChsdR mcrA mcrB mrr. Although no such strainhas been made, a recD sbcC hsdR mcrA mcrBstrain, DL491, has been reported recently(Whittaker et al., 1988).

LITERATURE CITEDAppleyard, R.K. 1954. Segregation of new lyso-

genic types during growth of a doubly lysogenicstrain derived from Escherichia coli K12. Genet-ics 39:440-452.

Bachmann, B.J. 1983. Linkage map of Escherichiacoli K-12, edition 7. Microbiol. Rev. 47:180-230.

Backman, K., Ptashne, M., and Gilbert, W. 1976.Proc. Natl. Acad. Sci. U.S.A. 73:4174-4178.

Barkley, M.D. and Bourgeois, S. 1978. Repressorrecognition of operator and effectors. In TheOperon (J. Miller, ed.) pp. 177-220. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

Bassett, C.L. and Kushner, S.R. 1984. ExonucleasesI, III and V are required for stability of ColE1-related plasmids in Escherichia coli. J. Bacteriol.157:661-664.

Bickle, T. 1982. ATP-dependent restriction endonu-cleases. In Nucleases (S.M. Linn and R.G.Roberts, eds.) p. 85. Cold Spring Harbor Labo-ratory, Cold Spring Harbor, N.Y.

Biek, D.P. and Cohen, S.N. 1986. Identification andcharacterization of recD, a gene affecting plas-mid maintenance and recombination in Es-cherichia coli. J. Bacteriol. 167:594-603.

Bolivar, F. Rodriguez, R.L., Greene, T.J., Betlach,M.C., Heyneker, H.L., Boyer, H.W. 1977. Con-struction and characterization of new cloningvehicles. II. A multipurpose cloning system.Gene 2:95-113.

Borck, J., Beggs, J.D., Brammer, W.J., Hopkins,A.S., and Murray, N.E. 1976. The constructionin vitro of transducing derivatives of phagelambda. Mol. Gen. Genet. 146:199-207.

Boyer, H.W. and Roulland-Dussoix, D. 1969. Acomplementation analysis of the restriction andmodification of DNA in Escherichia coli. J. Mol.Biol. 41:459-472.

Casadaban, M.J. and Cohen, S.N. 1980. Analysis ofgene control signals by DNA fusion and cloningin Escherichia coli. J. Mol. Biol. 138:179-207.

Celis, J.E. and Smith, J.D. 1979. Nonsense Muta-tions and tRNA Suppressors. Academic Press,London.

Chalker, A.F., Leach, D.R.F., and Lloyd, R.G. 1988.Escherichia coli sbcC mutants permit stablepropagation of DNA replicons containing a longpalindrome. Gene 71:201-205.

Demerec, M., Adelberg, E.A., Clark, A.J., and Hart-man, P.E. 1966. A proposal for uniform nomen-clature in bacterial genetics. Genetics 54:61-66.

Ehrlich, M. and Wang, R.Y. 1981. 5-methylcytosinein eukaryotic DNA. Science 212:1350-1357.


1.4.12


Bacterial Genetics

Foster, T.J. 1983. Plasmid-determined resistance toantimicrobial drugs and toxic metal ions in bac-teria. Microbiol. Rev. 47:361-409.

Frischauf, A-M., Lehrach, H., Poustka, A., and Mur-ray, N. 1983. Lambda replacement vectors car-rying polylinker sequences. J. Mol. Biol.170:827-842.

Gottlieb, D. and Shaw, P.D. 1967. Antibiotics. I.Mechanism of Action. Springer-Verlag, NewYork.

Hanahan, D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol.166:557-580.

Heitman, J. and Model, P. 1987. Site-specific methy-lases induce the SOS DNA repair response inEscherichia coli. J. Bacteriol. 169:3243-3250.

Huynh, T.V., Young, R.A., and Davis, R.W. 1985.Construction and screening of cDNA libraries inλgt10 and λgt11. In DNA Cloning, Vol 1: Apractical approach (D.M. Glover, ed.) pp 49-78.IRL Press, Oxford.

Joyce, C.M. and Grindley, N.D.F. 1984. Method fordetermining whether a gene of Escherichia coliis essential: Application to the polA gene. J.Bacteriol. 158:636.

Karn, J., Brenner, S., Barnett, L., and Cesareni, G.1980. Novel bacteriophage λ cloning vector.Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176.

Kunkel, T.A. 1987. Rapid and efficient site-specificmutagenesis without phenotypic selection.Methods Enzymol. 154:367-383.

Leach, D.R.F. and Stahl, F.W. 1983. Viability of λphages carrying a perfect palindrome in the ab-sence of recombination nucleases. Nature305:448.

Lewendon, A., Ellis, J., Shaw, W.V. 1995. Structuraland mechanistic studies of galactoside acetyl-transferase, the Escherichia coli LacA geneproduct. J. Biol. Chem. 270:26326-31.

Lindahl, G. and Sunshine, M. 1972. Excision-defi-cient mutants of bateriophage P2. Virology49:180-187.

Lloyd, R.G. and Buckman, C. 1985. Identificationand genetic analysis of sbcC mutations in com-monly used recBC sbcB strains of Escherichiacoli K-12. J. Bacteriol. 164:836-844.

MacNeil, D. 1988. Characterization of a uniquemethyl-specific restriction system in Streptomy-ces avermitilis. J. Bacteriol. 170:5607-5612.

Marinus, M.G. 1987. DNA methylation in Es-cherichia coli. Ann. Rev. Genet. 21:113-132.

Miller, J. 1972. Experiments in Molecular Genetics.Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

Miller, J.H. 1978. The lacI gene: Its role in lacoperon control and its uses as a genetic system.In The Operon (J. Miller, ed.) pp. 31-88. ColdSpring Harbor Laboratory, Cold Spring Harbor,N.Y.

Miller, J.H., Lebkowski, J.S., Greisen, K.S., andCalos, M.P. 1984. Specificity of mutations in-duced in transfected DNA by mammalian cells.EMBO J. 3:3117-3121.

Moazed, D. and Noller, H.F. 1987. Interaction ofantibiotics with functional sites in 16S ribosomalRNA. Nature 327:389-394.

Raleigh, E.A. and Wilson, G. 1986. Escherichiacoli K-12 restricts DNA containing 5-methyl-cytosine. Proc. Natl. Acad. Sci. U.S.A.83:9070-9074.

Raleigh, E.A., Murray, N.E., Revel, H., Blumenthal,R.M., Westaway, D., Reith, A.D., Rigby, P.W.J.,Elhai, J., and Hanahan, D. 1988. McrA and McrBrestriction phenotypes of some E. coli strains andimplications for gene cloning. Nucl. Acids Res.16:1563-1575.

Russel, M. and Model, P. 1984. Replacement of theflp gene of Escherichia coli by an inactive genecloned on a plasmid. J. Bacteriol. 159:1034-1039.

Silberstein, Z. and Cohen, A. 1987. Synthesis oflinear multimers of oriC and pBR322 derivativesin Escherichia coli K-12: Role of recombinationand replication functions. J. Bacteriol.169:3131-3137.

Stahl, F.W. 1986. Roles of double-strand breaks ingeneralized genetic recombination. In Progressin Nucleic Acid Research and Molecular Biol-ogy, Vol. 33 (W.E. Cohn and K. Moldave, eds.)pp. 169-194. Academic Press, San Diego.

Ullman, A., Jacob, F., and Monod, J. 1967. Charac-terization by in vitro complementation of a pep-tide corresponding to an operator-proximal seg-ment of the β-galactosidase structural gene ofEscherichia coli. J. Mol. Biol. 24:339-343.

Wang, X.G., Olsen, L.R., Roderick, S.L. 2002.Structure of the lac operon galactoside acetyl-transferase. Structure 10:581-588.

Wertman, K.F., Wyman, A.R., and Botstein, D.1986. Host/vector interactions which affect theviability of recombinant phage lambda clones.Gene 49:253-262.

Whittaker, P.A., Campbell, A.J.B., Southern, E.M.,and Murray, N.E. 1988. Enhanced recovery andrestriction mapping of DNA fragments cloned ina new λ vector. Nucl. Acids Res. 16:6725-6736.

Woodcock, D.M., Crowther, P.J., Diver, W.P., Gra-ham, M.W., Bateman, C., Baker, D.J., and Smith,S.S. 1988. RglB facilitated cloning of highlymethylated eukaryotic DNA: The human L1transposon, plant DNA and DNA methylation invitro with human DNA methyltransferase. Nucl.Acids Res. 16:4465-4482.

Woodcock, D.M., Crowther, P.J., Doherty, J., Jeffer-son, S., De Cruz, E., Noyer-Weidner, M., Smith,S.S., Michael, M.Z., and Graham, M.W. 1989.Quantitative evaluation of Escherichia coli hoststrains for tolerance to cytosine methylation inplasmid and phage recombinants. Nucl. AcidsRes. 17:3469-3478.


1.4.13


Wyman, A.R., Wertman, K.F., Barker, D., Helms, C.,and Petri, W.H. 1986. Factors which equalize therepresentation of genome segments in recombi-nant libraries. Gene 49:263-271.

Yanisch-Perron, C., Vieira, J., and Messing, J. 1985.Improved M13 phage cloning vectors and hoststrains: Nucleotide sequences of the M13mp18and pUC19 vectors. Gene 33:103-119.

Young, R.A. and Davis, R.W. 1983. Efficient isola-tion of genes by using antibody probes. Proc.Natl. Acad. Sci. U.S.A. 80:1194-1198.

Zabin, I. and Fowler, A.V. 1978. β-galactosidase, thelactose permease protein, and thiogalactosidetransacetylase. In The Operon (J. Miller, ed.) pp.89-122. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

KEY REFERENCESBeckwith, J. and Zipser, D., eds. 1970. The Lactose

Operon. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

An introduction to early work with the lactose op-eron.

Miller, 1972. See above.

Contains excellent introductions to experimentaltechniques for working with E. coli and λ-derivedphages.

Miller, J., ed. 1981. The Operon. Cold Spring Har-bor Laboratory, Cold Spring Harbor, N.Y.

An updated and thorough introduction to regulatorymechanisms, starting with the lactose operon.

Neidhardt, F.C., Ingraham, J.L., Low, K.B., Ma-gasanik, B., Schaechter, M., and Umbarger,H.E., eds. 1996. Escherichia coli and Salmonellatyphimurium: Cellular and Molecular Biology.American Society for Microbiology, Washing-ton, D.C.

Encyclopedic coverage of the biology of these usefulbacteria.

Ullman, A. and Perrin, D. 1970. Complementationin β-galactosidase. In The Lactose Operon (J.Beckwith and D. Zipser, eds.) pp. 143-172. ColdSpring Harbor Laboratory, Cold Spring Harbor,N.Y.

Definitive review of alpha-complementation.

Contributed by Elisabeth A. RaleighNew England BiolabsBeverly, Massachusetts

Karen ElbingClark & Elbing LLPBoston, Massachusetts



1.4.14


Bacterial Genetics

SECTION IIVECTORS DERIVED FROM PLASMIDS

UNIT 1.5Introduction to Plasmid BiologyPlasmids are self-replicating, extrachro-

mosomal DNA molecules found in virtually allbacterial species. In nature, plasmids occur inexuberant profusion, varying in structure, size,mode of replication, number of copies per bac-terial cell, ability to propagate in different bac-teria, transferability between bacterial species,and perhaps most important, in the traits theycarry. Most prokaryotic plasmids are double-stranded circular DNA molecules; however,linear plasmids have been identified in bothgram-positive and gram-negative bacteria. Thesize of plasmids varies widely, from severalkilobases to hundreds of kilobases. Replicationof plasmids depends on host-cell proteins butalso may require plasmid-encoded functions.Plasmid replication may be synchronized withthe bacterial cell cycle, resulting in a low num-ber of plasmid molecules per bacterial cell, orindependent of the host cell cycle, allowing forthe proliferation of hundreds of plasmid copiesper cell. Some plasmids freely transfer theirDNA across bacterial species, some only trans-fer their DNA into bacteria of the same species,and some do not transfer their DNA at all.Plasmids carry genes that specify a wide varietyof functions including: resistance to antibiotics,resistance to heavy metals, sensitivity to mu-tagens, sensitivity or resistance to bacterio-phages, production of restriction enzymes, pro-duction of rare amino acids, production of tox-ins, determination of virulence, catabolism ofcomplicated organic molecules, ability to formsymbiotic relationships, and ability to transferDNA across kingdoms.

Starting in the 1970s, vectors for propaga-tion, manipulation, and delivery of specificDNA sequences were constructed with frag-ments from naturally occurring plasmids, pri-marily Escherichia coli plasmids. All plasmidvectors contain three common features: a rep-licator, a selectable marker, and a cloning site.The replicator is a stretch of DNA that containsthe site at which DNA replication begins (theorigin of replication or ori), and that also in-cludes genes encoding whatever plasmid-en-coded RNAs and proteins are necessary forreplication. The selectable marker, necessaryfor following and maintaining the presence ofthe plasmid in cells, is usually dominant and isusually a gene encoding resistance to some

antibiotic. The cloning site is a restriction en-donuclease cleavage site into which foreignDNA can be inserted without interfering withthe plasmid’s ability to replicate or to conferthe selectable phenotype on its host. Over theyears, plasmid vectors have increased in so-phistication; in addition to these three basicelements many vectors now include featuresthat make them particularly suitable for specifictypes of experiments.

UNITS 1.6 & 1.7 describe procedures for makingplasmid DNA. The process by which plasmidsare introduced into E. coli is called transfor-mation. Transformation protocols are given inUNIT 1.8. For a list of vectors and their salientfeatures see APPENDIX 5.

REPLICATORSOne of the ways that replicators are classi-

fied is based on the number of plasmid mole-cules maintained per bacterial cell under someset of standard growth conditions, the so-calledcopy number of the plasmid. This book defineshigh-copy-number plasmids as those whichexist in ≥20 copies per bacterial cell grown inliquid LB medium, and low-copy-numberplasmids as those which exist in <20 copies percell. High-copy-number-plasmids tend to besmaller than low-copy-number plasmids. Theyare more commonly used in molecular biologi-cal techniques since it is easier to prepare largequantities of pure plasmid DNA from cells thatbear them. Low-copy-number plasmids areutilized when it is important to control the genedosage of a cloned sequence, for example whenthe DNA sequence or the protein it encodesmakes the host organism sick (see Table 1.5.1).

High-copy-number plasmids tend to be un-der relaxed control of replication. These re-laxed plasmids initiate DNA replication in aprocess controlled by plasmid-encoded func-tions (see Mechanism of Replication and Copy-Number Control), and replication does not de-pend on the unstable host replication initiationproteins synthesized at the start of the bacterialcell cycle. Because their replication dependsonly on the stable host enzymatic replicationmachinery, the copy number of these plasmidscan be greatly increased, or amplified, by treat-ment of the plasmid-containing cells with pro-tein synthesis inhibitors such as chlorampheni-

Supplement 41

Contributed by Rhonda FeinbaumCurrent Protocols in Molecular Biology (1998) 1.5.1-1.5.17Copyright © 1998 by John Wiley & Sons, Inc.

1.5.1


col or spectinomycin. These protein synthesisinhibitors prevent synthesis of the replication-initiation proteins required for chromosomalreplication, allowing the replication enzymesto be commandeered for plasmid replication.High-copy-number plasmids usually do nothave any mechanism to ensure correct segrega-tion of the plasmids to the daughter cells; theyrely on random assortment to partition at leastone copy of the plasmid to each daughter.

Low-copy-number plasmids are usually un-der stringent control. Initiation of replicationof these plasmids depends on unstable proteinssynthesized at the start of the bacterial cell cycleand thus is synchronized with the replication ofthe bacterial chromosome. As copy numberdecreases, random segregation of plasmid cop-ies is not sufficient to ensure that each daughtercell acquires a copy of the plasmid. However,most low-copy-number plasmids carry genesthat guarantee their maintenance in the bacte-rial population. Stabilizing loci that encode amechanism for postsegregational killing ofplasmid-free daughter cells and a system forefficiently resolving plasmid multimers so thatthey can be appropriately partitioned have beenidentified. In addition, cis- and trans-actingpartitioning loci (par) that are thought to con-stitute an active mechanism for distribution ofplasmid copies to daughter cells have also beenidentified.

MECHANISM OF REPLICATIONAND COPY-NUMBER CONTROL

While there are many different replicators,the majority of plasmid vectors used in routinerecombinant DNA work contain one of thefunctionally similar replicators derived fromplasmids ColE1 or pMB1 (for example, thepopular pBluescript series contain a ColE1 ori-gin, and the pUC plasmids are derived frompMB1). The ColE1 replicator is a 600-nucleo-tide DNA fragment that contains the origin ofreplication (ori), encodes an RNA primer, andencodes two negative regulators of replicationinitiation. All enzymatic functions for replica-tion of the plasmid are provided by the bacterialhost.

Plasmid replication begins with transcrip-tion of an RNA primer upstream of the ori(RNAII; see Fig. 1.5.1) by the host RNA po-lymerase. RNAII is elongated through and ter-minated downstream of the ori. Interaction ofa specific secondary structure in the nascentRNAII transcript with the DNA template re-sults in formation of a persistent hybrid be-tween RNAII and the DNA template such thatRNAII remains paired with the DNA templateat the ori. The RNAII transcript is cleaved byRNAseH at the ori sequence. This processedRNAII primer is extended by DNA polymeraseI to initiate plasmid replication. Regulation ofthe proper RNAII secondary structure controlsinitiation of DNA replication and is responsible

Table 1.5.1 Characteristics of Commonly Used Plasmid Replicators

Replicator Prototypeplasmid Size (bp) Markers on

prototypeCopya

number References

pMB1 pBR322 4,362 Apr, Tetr High; 100-300 Bolivar et al., 1977

ColE1 pMK16 ∼4,500 Kanr, Tetr, ColE1imm

High; >15 Kahn et al., 1979

p15A pACYC184 ∼4,000 Emlr, Tetr High; ∼15 Chang et al., 1978

pSC101 pLG338 ∼7,300 Kanr, Tetr Low; ∼6 Stoker et al., 1982

F pDF41 ∼12,800 TrpE Low; 1 to 2 Kahn et al., 1979

R6K pRK353 ∼11,100 TrpE Low; <15 Kahn et al., 1979

R1 (R1drd-17) pBEU50 ∼10,000 Apr, Tetr Low at 30°C; highabove 35°Cb

Uhlin et al., 1983

RK2 pRK2501 ∼11,100 Kanr, Tetr Low; 2 to 4 Kahn et al., 1979

λ dv λ dvgal —c Gal — Jackson et al., 1972

aCopy numbers are for the prototype plasmid. Plasmid vectors that contain replicators derived from these plasmids may have differentcopy numbers due to introduction of mutations into the replicator. For example, pUC series (pmB1-derived) has copy numbers of1000-3000.bTemperature sensitive.cNot known.


1.5.2

Introduction toPlasmid Biology

for determining the number of plasmid mole-cules per cell.

Both ColE1 and pMB1 plasmids are high-copy-number plasmids, maintained at between15 and 25 copies per bacterial cell, respectively.The copy number of these plasmids is regulatedby an antisense RNA transcript, RNAI, and theprotein ROP, the product of the rop gene. RNAIand the ROP protein act in concert to interceptformation of the proper RNAII secondary

structure. RNAI is exactly complementary tothe 5′ end of the RNAII transcript. RNAI basepairs with the 5′ end of RNAII, preventing theformation of the specific secondary structurenecessary for establishment of a persistent hy-brid between RNAII and the DNA template thatis a prerequisite for maturation of the RNAIIprimer. ROP protein stabilizes the interactionbetween the antisense RNAI and the primerRNAII. Mutations that disrupt or destabilize

RNAIIelongation of RNAII transcription

no binding of RNAI

RNAII

RNAII

RNAII

formation of persistent hybrid at oriori

ori

RNAI

RNAI/RNAII duplex

ori

ori

ori

RNase H cleavage

initiation of DNA leading-strand synthesis

no persistent hybrid formed at ori

no primer maturation

binding of RNAI

ori

RNAII (preprimer)

–

RNAI

formation of RNA primer

ori

Figure 1.5.1 ColE1 replication initiation and copy number control. ColE1 replication requires theformation of a RNA primer to initiate DNA synthesis. The RNA primer is derived from processingof a transcript, RNAII (bold), that starts upstream of the ori. Appropriate processing of the RNAIItranscript is dependent on formation of a persistent hybrid between RNAII and the ori DNA template.If the proper RNAII secondary structure forms, then the RNA/DNA duplex is maintained at the oriand RNAse H can cleave the RNAII transcript to generate the primer for DNA synthesis. Processingof RNAII to form the primer is regulated by a second transcript, RNAI. RNAI is complementary tothe 5′ end of RNAII. If the RNAI transcript forms a duplex with RNAII, RNAII cannot take on thesecondary structure necessary to create the persistent hybrid at the ori and maturation of the RNAIIprimer is inhibited. The copy number of ColE1 plasmids is determined by the balance betweensuccessful RNAII processing events and those inhibited by RNAI. Adapted from Gerhart et al. (1994)with permission from the Annual Review of Microbiology.


1.5.3


the RNAI/RNAII interaction result in a higherplasmid copy number. For example, the pUCplasmid series have pMB1 replicators that donot contain an intact rop gene, which accountsfor a 2-fold increase in copy number over plas-mids with intact pMB1 replicons. Interestingly,additional mutation(s) in the origin are the mainfactor in the ultra-high-copy number (1000 to3000) of this plasmid series (K. Struhl, unpub.observ.)

PLASMID INCOMPATIBILITYFor experiments that require that more than

one plasmid vector be maintained in a bacterialcell at the same time, another critical feature ofplasmid replicators is whether or not two plas-mid replicators are compatible. Two plasmidsare said to be incompatible with one another,and hence belong to the same incompatibilitygroup, if they cannot stably co-exist. Plasmidsare generally incompatible if they share anyfunction required for the regulation of plasmidreplication. For example, ColE1- and pMB1-derived plasmids are incompatible with oneanother but are compatible with p15A plas-mids. The incompatibility of ColE1 and pMB1plasmids is a consequence of two facts: first,that plasmid DNA replication of these plasmidsis negatively controlled by RNAI which acts intrans on other plasmids with the same primerRNA, and second, that these plasmids lack amechanism to ensure that each plasmid in a cellreplicates once per cell cycle. Therefore, if acell that contains a pMBI-derived plasmid issubsequently transformed with a ColE1-de-rived plasmid, cells selected to contain theColE1 plasmid will usually have lost the pMBIplasmid.

SELECTABLE MARKERSIn order to guarantee that a plasmid vector

is taken up by and maintained in bacterial cells,there must be a way to select for plasmid-con-taining cells. Genes encoding resistance to an-tibiotics such as ampicillin, tetracycline,kanamycin and chloramphenicol are the mostcommon bacterial selectable markers for plas-mid vectors. Typically, cells are transformedwith plasmid DNA using the technique de-scribed in UNIT 1.8 and then plated out on LBplates that contain the proper antibiotic (seerecipes in UNIT 1.1). Only the bacterial cellscontaining the plasmid will grow on the selec-tive medium; the antibiotic-resistance pheno-type conferred is dominant to the antibiotic-sensitive phenotype of cells that do not possessthe plasmid vector. Another dominant select-

able marker that is occasionally used is theimmunity to infection by phage lambda(lambda repressor). In addition, recessivemarkers are sometimes used in plasmid selec-tions; for example, leuB− E. coli cannot growin the absence of leucine, and selection forgrowth of these strains in the absence of leucineallows isolation of colonies transformed with aplasmid that contains a gene that complementsleuB.

For experiments that involve introduction ofplasmid vectors into systems other than bacte-ria, such as yeast or mammalian cells, it isusually necessary to select for the presence ofplasmid DNA in these hosts. Selectable mark-ers for yeast and mammalian systems are gen-erally distinct from those used in E. coli (seePlasmid Vectors for Yeast and Plasmid Vectorsfor Expression in Cultured Mammalian Cells).In cases where a vector is needed that will beused in both E. coli and another host, it isnecessary for the plasmid vector to carry select-able markers for both hosts.

CLONING SITEToday’s plasmid vectors contain a multiple

cloning site (MCS) or polylinker cloning re-gion that can include >20 tandemly arrangedrestriction endonuclease sites. The sites in thepolylinker are almost always designed to beunique within the vector sequence so that cut-ting the vector with a restriction endonucleasein the polylinker and cloning foreign DNA intothis site does not disrupt other critical featuresof the vector. The plethora of sites in thepolylinker ensures that the appropriate enzymesites will be available for cloning most DNAfragments, provides unique reference restric-tion sites for rapid restriction mapping of theinsert, and generally allows for a great deal offlexibility when manipulating the cloned DNA.

The sequences that directly flank thepolylinker site are often useful for manipulationor analysis of insert DNA. Many polylinkersites are flanked by sequences for which thereare commercially available complementary oli-gonucleotides, for example the M13 reverse,−20, and −40 primers, that can be used forpriming polymerase chain reactions (PCR) orDNA sequencing reactions. Such primers areuseful tools for amplification or sequencing ofany DNA fragment inserted into the polylinker.Some polylinkers are bordered by 8-bp-cutterrestriction sites, like Not1. These sites occurinfrequently in DNA and thus allow for the easyexcision of an intact insert fragment from theplasmid vector.


1.5.4


Many plasmid vectors have been developedwith bacteriophage—SP6, T7, or T3—promot-ers flanking the polylinker cloning sites. Thesepromoters can be used in vitro or in vivo, if thebacteriophage RNA polymerase is also present,to produce large quantities of RNA transcriptsfrom DNA inserted into the polylinker.

In order to make it easier to identify plas-mids that contain insert DNA, the polylinkersof some vectors have been engineered so thatintroduction of DNA into the polylinker resultsin a scorable phenotype. The most commonexample of this is insertion of the polylinker ofmany basic cloning vectors, the pUC series forexample, into the lacZ gene α fragment. In theappropriate genetic background, production ofthe lacZ α fragment allows for formation of anactive β-galactosidase enzyme which results inthe formation of blue colonies on Xgal/IPTGindicator plates. Cloning into these polylinkersprevents production of a functional lacZ αfragment, allowing for rapid identification ofplasmids containing inserts as white colonieson Xgal/IPTG indicator plates. Vectors havealso been developed that allow for direct selec-tion of plasmids containing inserts by locationof the polylinker in the ccdB (control of celldeath) gene which causes cell death in E. coli.Disruption of the ccdB gene by introduction ofan insert into the polylinker allows the cells tosurvive, and thus only recombinants will growunder conditions where the ccdB gene is ex-pressed.

CHOOSING A PLASMID VECTORSome of the basic features to consider when

selecting a plasmid vector include the size ofthe vector, its copy number, the polylinker, andthe ability to select and/or screen for inserts.Large plasmids, >15 kb, do not transform welland frequently give lower DNA yields. Con-sider the final size of the vector plus insert whenplanning an experiment and wherever possibleuse smaller vectors. The higher the copy num-ber the more vector DNA is produced, buthigh-copy-number vectors may not be applica-ble to all situations (see Mechanism of Repli-cation and Copy-Number Control). Whenchoosing a plasmid vector, consider both whatsites are present in the polylinker and the orderof the sites. If it will be necessary to manipulatethe cloned sequence subsequent to insertioninto the polylinker, plan ahead to ensure thatthe necessary sites will remain available in thepolylinker. Selections or screens for identifica-tion of recombinant clones are useful for ex-periments where cloning efficiency is expected

to be low or when generation of a large numberof clones is necessary. However, for routinesubcloning experiments the advent of PCR(UNIT 15.1) has made it possible to rapidly screenthrough a large number of transformants toidentify potential recombinant molecules, ob-viating the need for histochemical and geneticscreening methods.

The primary factor in choosing a plasmidvector is to understand and anticipate the ex-periments for which the recombinant clone willbe used. The specialized functions of plasmidvectors are generally the key to selecting thecorrect vector for an experiment. For example,completely different types of vectors would beused for generating large quantities of DNA,expressing a fusion protein in bacteria, or forundertaking a two-hybrid screen in yeast. Oncethe type of vector required is determined thendeciding upon a particular vector is dependenton both the details of the ancillary vector fea-tures, for example the type of promoter used toexpress a recombinant protein, and the proper-ties of the replicator, polylinker, and selectablemarker.

PLASMID VECTORS FORPRODUCTION OFSINGLE-STRANDED DNA

Plasmids have been developed that containa filamentous phage origin of replication inaddition to a plasmid ori. These “phagemid”vectors (UNIT 1.14) can be grown and propagatedas plasmids. However, upon super-infection ofa plasmid-containing cell with a wild-typehelper phage, the phage ori becomes active, andsingle-stranded DNA (ssDNA) is produced andsecreted. There are usually (+) and (−) versionsof these vectors where the phage ori is inopposite orientation so that it is possible toproduce ssDNA from either DNA strand. Formany years, ssDNA was the substrate of choicefor DNA sequencing (UNIT 7.4) and oligonu-cleotide-directed mutagenesis (UNIT 8.1). Devel-opment of sequencing and mutagenic protocolsthat use double-stranded templates has madethe production of ssDNA a less frequently util-ized feature of plasmid vectors. ThepBluescriptI, pBluescriptII, and pBS phagemidvectors derived from the general cloning vectorpUC19 (Fig. 1.5.2) are examples of phagemidsthat incorporate the f1 filamentous phage ori.

PLASMID VECTORS FORCLONING LARGE INSERTS

Cosmid vectors, plasmids carrying a lambdaphage cos site (e.g., pWE15, Fig. 1.5.3), were


1.5.5


pUC192686 bp

Apr

lac Z′

Plac

′lac l

ori

AGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCAT

EcoRISacIKpnISmaI XmaIBamHIXbaIHincIIPst ISphIHindIII

396

447

Nar l 235Nde l 183

Eco O 109 2674

Aat lI 2617Ssp l 2501

Xmn l 2294

Scal 2177

Gsu l 1784

Cfr10 I 1779PpaI 1766

AfI III 806

EcoRI

400

Sac I KpnISmaIXmaI

BamHI XbaI HincIIAcc ISal I

Pst I SphI HindIII

420 440 460

lac Z′ ThrlleMetThr(Met)1

Figure 1.5.2 Map of pUC19.

T7T3

Eco

RI

Not

I

Bam

HI

Not

I

Eco

RI

Apr

ori

cos

Neor

SV40 ori

pWE158.2 kb

Figure 1.5.3 Map of pWE15 (adapted from Wahl et al., 1987, with permission).


1.5.6


developed to facilitate cloning of large DNAfragments (UNIT 1.10). Cosmids can be trans-formed into cells like plasmids and once in thecells, replicate using their plasmid ori. TheColE1 type replicators are the most commonlyused in these vectors, and cosmids can gener-ally be maintained at high-copy-number in E.coli. Cosmids can also be packaged into lambdaphage heads. In order for packaging to occurthe cos sites must be separated by 40 to 50 kb,the approximate length of the wild-type lambdagenome. Many cosmid vectors are between 5to 10 kb in size and therefore can accept insertsof 30 to 45 kb. Because of the size of insertsthey can accept, the high efficiency of packag-ing recombinant molecules into phage, and theefficiency of infection of cosmid-containingphage heads, cosmid vectors are frequentlyused for making genomic libraries. Unfortu-nately, propagation of insert-containing cosmidvectors in E. coli sometimes results in the de-letion of all or a portion of the insert. To addressthis problem, a new set of cosmid vectors havebeen developed that replace the ColE1 replica-

tor with the F factor replicator. These “fosmid”vectors are maintained at low-copy-number, 1to 2 copies per cell, and are more stable thanhigher-copy-number cosmid vectors whengrown in E. coli (Kim et al., 1992).

Another type of plasmid cloning vector,called bacterial artificial chromosome (BAC;Fig. 1.5.4), has been developed using the Ffactor replicator for propagation of very largepieces of DNA (100 to 500 kb). The vectors areused in a similar manner to yeast artificialchromosome (YAC) vectors but have the ad-vantage of being manipulated solely in E. coli.

PLASMID VECTORS FOREXPRESSION OF LARGEQUANTITIES OF RECOMBINANTPROTEINS

There are a wide variety of vectors for ex-pressing high levels of recombinant proteins inE. coli, insect, and mammalian cells (Chapter16). The general goal of expressing proteins inany of these systems is to produce large quan-tities of a particular protein upon demand.

par B

par C

par A

pBeloBAC117.3 kb

SaII NotI H B NotI

cosN IoxP IacZ

SnaBI

EcoRV

EcoRV

rep E

ori S

CMr

EcoRI

XhoI

XbaI

T7 SP6

Figure 1.5.4 Map of pBeloBAC11 (adapted from Shizuya et al., 1992, with permission). Abbre-viation: CM, chloramphenicol.


1.5.7


While the features of the protein expressionsystems vary considerably, the basic propertiesoutlined for expressing proteins in E. coli arecommon to all of them. Expression vectors areusually designed such that production of theforeign protein is tightly regulated. This is nec-essary because the host cellular machinery isco-opted to produce large quantities of the for-eign protein, the shear amount of which maybe toxic to the cell, and/or the foreign proteinmay encode a function that will inhibit cellgrowth or kill the host cell. Generally, expres-sion vectors are configured so the polylinkercloning site is downstream of an inducible pro-moter. One of the promoters commonly used inE. coli expression vectors is the hybrid trp/lacpromoter (trc) which contains the lacO operatorsite. This promoter is turned off in the presenceof the lacIq repressor; the repressor gene iseither carried by the bacterial host or is encodedon the expression vector itself. Expression ofthe foreign protein from the trc promoter isinduced by the addition of IPTG.

The quantity of foreign protein producedwill be determined by both the rate of transcrip-tion of the gene and the efficiency of translationof the mRNA. Therefore, in addition to regu-lated highly inducible promoters, many E. coliprotein expression vectors are designed to op-timize translation of the foreign protein in bac-terial cells. These expression vectors include anefficient ribosome binding site and an ATG startcodon uptsream of the polylinker cloning site.Usually the cloning sites in the polylinker aredesigned so that it is possible to make an in-frame fusion to the protein of interest in all threereading frames.

For many experiments, high levels of purerecombinant protein are required, and someexpression vectors are designed to createtagged or fusion proteins that facilitate purifi-cation of the recombinant protein. Tag se-quences may be located 5′ or 3′ to the poly-linker, creating either amino- or carboxy-termi-nal-tagged fusion proteins. Six polyhistidineresidues (UNIT 10.11B), the FLAG epitope, andglutathione-S-transferase protein (UNIT 16.7) aresome of the sequences that are appended toproteins to assist in purification. Tagged orfusion proteins can be rapidly and efficientlypurified using an appropriate affinity columndesigned to tightly bind the tag or fusion region.Many protein expression vectors are createdwith specific protein cleavage sites adjacent tothe tag or fusion sequences to allow for removalof these sequences from the purified protein.This feature may prove to be essential if the tag

or fusion sequence impairs the function of theprotein in the relevant assays.

PLASMID VECTORS FORREPORTER GENE FUSIONS

Plasmid vectors have been designed to sim-plify the construction and manipulation of re-porter gene fusions, where a promoter of inter-est is used to drive an easily scored marker gene(UNIT 9.6). Gene fusions provide a rapid andsimple method for following the expressionpattern conferred by a particular promoter.There are generally two types of reporter fu-sions, transcriptional and translational fusions.For transcriptional fusions, the ATG start codonis provided by the marker gene. In translationalfusions, the 5′ untranslated region and ATG areprovided by the gene of interest; in fact theseconstructs may fuse a large portion, or evenentire coding region, to the amino terminus ofa marker. In the reporter vector, the polylinkercloning site is located directly upstream of thereporter gene for insertion of the promoterfragment. In vectors that are designed for ex-pression in eukaryotic cells, a polyadenylationsignal is located downstream of the reportergene. There are a variety of reporter genes usedincluding chloramphenicol acetyltransferase,luciferase, β-galactosidase, secreted alkalinephosphatase, human growth hormone, β-glu-curonidase, and green fluorescent protein (alsosee UNITS 9.6-9.7C). An example of a reportervector pEGFP, is shown in Figure 1.5.5. Thechoice of reporter gene will depend on the celltype or organism, whether the assay will bedone in vivo or in vitro, and whether quantita-tive or qualitative data is desired. The replicator,selectable marker, and other elements of thereporter vector will also have to be compatiblewith the system.

PLASMID VECTORS FOR YEASTYeast plasmid vectors contain the same ba-

sic features as E. coli vectors—replicator, se-lectable marker, and cloning site (UNIT 13.4).There are two primary types of replicators usedin yeast plasmid vectors, autonomously repli-cating sequences (ARS) derived from the yeastchromosomes and the natural yeast 2µm plas-mid replicator. ARS-containing plasmids fre-quently contain yeast centromeric sequences toensure their stable maintenance in the popula-tion of cells. Both ARS and 2µm vectors aver-age 10 to 30 copies per cell. Many yeast vectorsare “shuttle” vectors that can be maintained inboth S. cerevisae and E. coli (e.g., pRS303; seeFig. 1.5.6). These vectors have an E. coli plas-


1.5.8


mid replicator, frequently pMB1 derived, anda yeast replicator. Conversely, the yeast inte-grating plasmid vectors, used for introductionof genes into the yeast chromosome, have abacterial replicator but no yeast replicator.

The selectable markers in yeast are for themost part recessive markers, usually clonedyeast genes that are used to complement muta-

tions in a biosynthetic pathway. For example,the URA3 marker carried on a plasmid is usedto restore the ability to grow in the absence ofuracil to a ura3− yeast. Therefore, yeast select-able markers, unlike most bacterial selectablemarkers, are strain dependent.

There are a wide variety of yeast vectorsavailable that have a range of specialized func-

pE

GF

P-1

4.2

kb

MC

S

Not

I

EG

FP

pUC

ori

HS

V T

Kpo

ly A

Kan

r /

Neo

rS

V40

ori

PS

V40

e

SV

40po

ly A

f1 ori

P

MC

S

TA

GC

G C

TA

CC

G G

AC

TC

A G

AT

CT

C G

AG

CT

C A

AG

CT

T C

GA

AT

T C

TG

CA

G T

CG

AC

G G

TA

CC

G C

GG

GC

C C

GG

GA

T C

CA

CC

G G

TC

GC

C A

CC

AT

G G

TG

Eco

47III

Bgl

IIX

hoI

Sac

IE

cl13

6II

Eco

RI

Pst

IS

alI

Acc

IK

pnI

Asp

7181

Sac

IIA

paI

Bsp

1201

Xm

aI

Sm

aIB

amH

IA

geI

EG

FP

Hin

dIII

Fig

ure

1.5

.5

Map

of p

EG

FP

-1.


1.5.9


tions—e.g., expression of recombinant pro-teins in yeast, integration of sequences into theyeast genome, and cloning of very large frag-ments (hundreds of kilobases) of genomicDNA.

PLASMID VECTORS FOREXPRESSION IN CULTUREDMAMMALIAN CELLS

The type of vector that is used in mammaliancells depends on whether the experiment in-volves transient transfection into mammaliancells or the generation of stable mammalian celllines carrying the construct of interest (UNITS

9.1-9.5). Virtually any plasmid vector that con-tains an appropriate construct for expression inmammalian cells can be used in transient as-says. In transient assays, the plasmid vectorcarrying the DNA of interest is transfected intomammalian cells, the cells are harvested sometime later (24 to 96 hr), and the pertinent assay

is performed. It is not necessary for plasmidvectors used in these assays to carry a mammal-ian selectable marker or to replicate in mam-malian cells; therefore easily manipulatablebacterial plasmids, like pUC, are usually thevectors of choice.

Plasmid vectors carrying a selectablemarker that functions in mammalian cells arenecessary for the generation of stable trans-genic lines (UNITS 9.5; e.g., pcDNA3.1, Fig.1.5.7). In order to generate a stable mammaliancell line, the plasmid DNA is transfected intothe mammalian cells, and over a period ofseveral weeks the DNA of interest is selectedfor based on expression of the vector-bornemarker. Many mammalian vectors cannot rep-licate in mammalian cells, and the only way tomaintain the DNA of interest and the selectablemarker is for the vector to randomly integrateinto the mammalian genome. However, thereare some plasmid vectors that carry the simian

SacI

2175

CEN6

ARSH4 pRS3134967

AatII 4384

HindIII 808

BstXI 886

HindIII 995

KpnI 1122

PstI 1183

NaeI 1748PvuI 1920

PvuII1950

ScaI 3944

PvuI 3833

PvuII 2395

KpnI XhoI ClaI EcoRV Pst I BamHI XbaI EagI BstXI

ApaI SaII HindIII EcoRI SmaI SpeI NotI SacII

T3 T7

2073

1

2

3

0

ori lacZ

HIS3

pRS3034453 bp

f1(+)

4Apr

Figure 1.5.6 Map of pRS303 (adapted from Sikorski and Heiter, 1989, with permission).


1.5.10


virus (SV40) or bovine papilloma virus (BPV)ori that can replicate in mammalian cells if thenecessary viral replication proteins are pro-vided; in most cases the viral replication pro-teins must be provided by the host cell line,limiting the range of cell types in which thesevectors are useful.

The most critical general feature of plasmidvectors (and also viral vectors) for creation ofstable mammalian cell lines is the selectablemarker (see UNIT 9.5). Unlike transient assays,formation and maintenance of stable cell linesrequires selection. The number of selectablemarkers available for mammalian cells is lim-ited; resistance to hygromycin, puromycin,G418, and neomycin are the predominantmarkers used. Since expression of foreign pro-teins or assaying the expression of a mammal-ian gene may require multiple plasmids and/orintegration of constructs into the mammalianchromosome, careful planning must take placeto ensure that all constructs can be selected forwith the limited number markers. Further, sinceselection may be necessary over a long period

of time—weeks for generation of the lines andyears for their maintenance—it is important torecognize that some of the antibiotics used inselection are very expensive and thus cost maybe a factor in experimental design.

Specialized plasmid vectors are also usedfor production of viruses that can infect mam-malian cells. Plasmid vectors have been de-signed to produce infectious retroviral particleswhen transfected into the appropriate packag-ing cell line (UNIT 9.9). These retroviral vectorsare then used to create stable transgenic linesin mammalian cell types not amenable to trans-fection. In addition, plasmid vectors have beendesigned to allow easy insertion of DNA se-quences into vaccinia virus for the purpose ofcreating recombinant viruses that overexpressrecombinant proteins (UNIT 16.15).

PLASMID VECTORS FORNON–E. COLI BACTERIA

Three features required of all bacterial plas-mid vectors are that they replicate (unless theyare suicide vectors), carry a selectable marker,

Hin

dIII

Eco

RV

Not

I

Xba

IA

paI

Nhe

IP

meI

Afl

IIH

indI

IIA

sp71

8IK

pnI

Bam

HI

Bst

XI

Eco

RI

Eco

RV

Bst

XI

Not

IX

hoI

Xba

IA

paI

Pm

eI

T7

PCMVBGH polyA

f1 ori

SV40 ori

Apr

Neor Zeoror

ColE1

SV40 polyA

pcDNA3.1Vectors

pcDNA3.1(+)

pcDNA3.1(–)

Nhe

IP

me

I

Eco

RI

Bst

XI

Pm

eI

T7

Xho

I

Bst

XI

Bam

HI

Asp

718I

Afl

II

Kpn

I

Figure 1.5.7 Map of pcDNA3.1.


1.5.11


and can be easily introduced into host cells. Thefirst thing to consider when selecting a plasmidvector for use in a non-E. coli host is whetheror not it can replicate and be stably maintainedin the particular strain. Different plasmid rep-licators have different host ranges, some havea narrow host range and can only replicate in aspecific strain while others are promiscuousand can replicate in a wide variety of host (e.g.,pRR54, Fig. 1.5.8). Unfortunately, the ColE1-type replicators have a narrow host range, thusmany standard E. coli vectors cannot be main-tained in other bacteria. However, a number ofbroad-host-range replicators, such as RK2 andRSF1010, have been well characterized andused to construct vectors that can replicate inmany gram-negative bacterial species (and inthe case of RSF1010, some gram-positive spe-cies as well).

Antibiotic resistance genes are used as se-lectable markers in non-E. coli bacterial hosts;however, the quantity of an antibiotic used toselect against non-plasmid-containing cells isusually higher than the quantity used for E. coliselection. Furthermore, some bacterial strainsare inherently resistant to particular antibiotics,thus it is important to determine whether theselectable marker carried on a plasmid vectoris functional in a particular strain.

The favored method for introduction of plas-mid DNA into bacterial host cells varies widelywith the bacterial strain. Unlike E. coli, manybacteria cannot be efficiently transformed bychemical procedures or electroporation. Inthese cases bacterial mating is used to introduceplasmid DNA into the desired bacterial host.Mating to transfer a plasmid vector from an E.coli host where the vector is maintained andmanipulated to a recipient bacterium requiresboth cis- and trans-acting functions. The tra (ormob) genes encode the trans-acting proteinsnecessary to transfer the plasmid DNA fromone bacterium to another, and they are usuallylocated on a helper plasmid that is distinct fromthe plasmid vector. Any plasmid vector that isto be mobilized must contain the cis-acting sitecalled oriT where the DNA is cleaved andtransfer is initiated.

MAPS OF PLASMIDSFigures 1.5.2-1.5.11 present maps of plas-

mids that are in widespread use or are examplesof plasmids whose special functions make themuseful for particular techniques described inthis manual. Note that the trend in developmentof vectors is to include multiple features on asingle vector, and many of these examples spanthe vector categories described.

trfAoriV

bla

ori T

par

pRR548.1 kb

H3 SphI XbaI BamHI

Figure 1.5.8 Map of pRR54 (adapted from Roberts et al., 1990, with permission).


1.5.12


pBR322 is one of the classic cloning vectorsfrom which many other vectors are derived. Itcontains an amplifiable pMB1 replicator andgenes encoding resistance to ampicillin andtetracycline. Insertion of DNA into a restrictionsite in either drug-resistance gene usually inac-tivates it and allows colonies bearing plasmidswith such insertions to be identified by theirinability to grow on medium with that antibiotic(see Fig. 1.5.9; Bolivar et al., 1977; sequencein Sutcliffe, 1978).

pUC19 belongs to a family of plasmid vec-tors that contains a polylinker inserted withinthe alpha region of the lacZ gene. The polylink-ers are the same as those used in the m13mpseries (Fig. 1.14.2). pUC19 and pUC18 havethe same polylinker but in opposite orienta-tions. Under appropriate conditions (see UNIT 1.4

for a description), colonies that bear plasmidscontaining a fragment inserted into thepolylinker form white colonies instead of blueones. These pMB1-derived plasmids (see Fig.1.5.2) maintain a very high-copy-number(1000 to 3000 per genome). Wild-type andrecombinant plasmids confer ampicillin resis-tance and can be amplified with chlorampheni-col (Norrander et al., 1983). In addition wild-type plasmids confer a LacZ+ phenotype toappropriate cells (e.g., JM101 cells, UNIT 1.4).

pBluescript is a commonly used phagemidcloning vector that contains a polylinker in-

serted into the alpha region of the lacZ gene andT3 and T7 promoter sequences flanking thecloning sites. The f1 (+) filamentous phageorigin of replication in pBluescript SK+ allowsfor the recovery of the sense strand of the lacZgene as ssDNA; the pBluescript SK(−) vector(Fig. 1.5.10) with the f1 origin in the oppositeorientation, f1(−), facilitates recovery of theother strand. The position of the polylinker inthe alpha region of the lacZ gene allows foridentification of inserts based on a blue/whitecolor screen under the appropriate conditions.The T3 and T7 promoters are recognized bybacteriophage RNA polymerases. Transcrip-tion from these promoters reads into thepolylinker from either side. RNA transcripts ofany DNA cloned into the polylinker can thusbe produced by run-off transcription in vitro.

pWE15 is an example of a cosmid vectorused for cloning DNA fragments ∼35 to 45 kb(see Fig. 1.5.3; Wahl et al., 1987). The cos sitesallow the DNA to be cut and packaged intophage heads by the appropriate lambda pro-teins. There is a single unique BamHI cloningsite flanked by T3 and T7 promoter sequences.These promoters are particularly useful for pro-duction of labeled RNA probes correspondingto the ends of the insert DNA, and these can beused to identify overlapping cosmids for chro-mosome walking and construction of cosmidcontigs. Not1 sites flanking the cloning site can

pBR3224363bp

Apr Tetr

ori

EcoRI 4361 ClaI 23HindIII 29

EcoRV 185

NheI 229BamHI 375

SphI 562Sal I 651

EagI 939NruI 972BspMI 1063

BsmI 1353Sty I 1369AvaI 1425

Bal I 1444BspMII 1664

PvuII 2066

Tth111I 2219

Nde l 2297

Afl lII 2475

PpaI 3435

PstI 3609PvuI 3735

ScaI 3846

SspI 4170

AatII 4286

Figure 1.5.9 Map of pBR322 (Bolivar et al. 1977; sequence in Sutcliffe, 1978).


1.5.13


pB

lues

crip

t S

K (

+/–)

2958

bp

Ssp

I 285

0S

spI 2

2

Nae

I 134

Ssp

I 445

Nae

I 333

Pvu

I 503

Pvu

II 53

2

Kpn

I 657

Sac

I 759

Pvu

II 97

7

Afl

III 1

153

Xm

nI 2

645

Sca

I 252

6

Pvu

I 241

6A

pr

f1(+

)ori

lacZ M

CS

Col

E1o

ri

reve

rse

prim

er5′

–G

GA

AA

CA

GC

TA

TG

AC

CA

TG

–3′

T3

prim

er5′

–A

AT

TA

AC

CC

TC

AC

TA

AA

GG

G–

3′S

K p

rimer

5′–

CG

CT

CT

AG

AA

CT

AG

TG

GA

TC–

3′

5′–

GG

AA

AC

AG

CT

AT

GA

CC

AT

GA

TT

AC

GC

CA

AG

CT

CG

AA

AT

TAA

CC

CT

CA

CT

AA

AG

GG

AA

CA

A

3′–

CC

TT

TG

TC

GA

TAC

TG

GTA

CT

AA

TG

CG

GT

TC

GA

GC

TT

TA

AT

TG

GG

AG

TG

AT

TT

CC

CT

TG

TT

ME

TT

3 pr

omot

er

+

1

816

β-G

alac

tosi

dase

AA

GC

TG

GA

GC

TC

CA

CC

GC

GG

TG

GC

GG

CC

GC

TC

TA

GA

AC

TA

GT

GG

AT

CC

CC

CG

GG

CT

GC

AG

GT

TC

GA

CC

TC

GA

GG

TGG

CG

CC

AC

CG

CC

GG

CG

AG

AT

CT

TG

AT

CA

CC

TA

GG

GG

GC

CC

GA

CG

TC

CT

TA

A

759

Sac

IS

acII

Bst

XI

Eag

IN

otI

Xba

IS

peI

Bam

HI

Pst

IE

coR

IS

ma

I

CC

AA

TT

CG

CC

CT

AT

AG

TG

AG

TC

GT

AT

TA

CA

AT

TC

AC

TG

GC

CG

TC

GT

TT

TAC

AA

–3′

(+)

GG

TT

AA

GC

GG

GA

TA

TC

AC

TC

AG

CA

TA

AT

GT

TA

AG

TG

AC

CG

GC

AG

CA

AA

AT

GT

T–

5′ (–

)

AA

TT

CG

AT

AT

CA

AG

CT

TA

TC

GA

TA

CC

GT

CG

AC

CT

CG

AG

GG

GG

GG

CC

CG

GTA

CC

GC

TA

TA

GT

TC

GA

AT

AG

CT

AT

GG

CA

GC

TG

GA

GC

TC

CC

CC

CC

GG

GC

CA

TG

G

3′–

CT

AT

GG

CA

GC

TG

GA

GC

T–5′

KS

prim

er3′

–C

GG

GA

TA

TCA

CT

CA

GC

AT

AA

TG

–5′

T7

prim

er3′

–TG

AC

CG

GC

AG

CA

AA

ATG

–5′

M13

20 p

rimer

Hin

dIII

Eco

RV

Bsp

106I

Cla

IS

alI

Acc

IX

hoI

Dra

II

Eco

010

9I

Apa

IK

pnI

657

+1

T

7 pr

omot

er

Hin

dII

Fig

ure

1.5

.10

Map

of p

Blu

escr

ipt S

K (

+/−)

.


1.5.14

potentially be used to excise an intact insertfragment from the vector. The ColE1-derivedori and ampicillin resistance gene allow forreplication and selection in bacteria. The SV40promoter (included in SV40 ori) which drivesthe neomycin phosphotransferase gene enablesselection in eukaryotic cells.

pBeloBAC11 is an example of the family ofbacterial-artificial-chromosome vectors basedon the low-copy-number F factor replicator(see Fig. 1.5.4; Shizuya et. al., 1992). BACvectors are used for cloning large DNA frag-ments (100 to 500 kb) in E. coli and are usedcommonly in genome-mapping strategies.oriS, repE, parA, parB, and parC genes are the

essential genes that compose the F factor rep-licator. oriS and repE genes are required forunidirectional replication of the plasmid, andparABC loci stably maintain the copy numberat one to two per E. coli genome. There are twounique cloning sites (HindIII and BamHI) in-serted into the lacZ alpha region. Other usefulfeatures of the cloning region are (1) T7 andSP6 promoter sequences flanking the cloningsites, (2) NotI restriction sites flanking the clon-ing sites for potential excision of the insert, and(3) and presence of the loxP and cosN sites thatcan be cleaved by specific enzymes. The endsgenerated by cleavage at loxP or cosN can beused as fixed reference points in building an

pTrc99A,B,C

T1

T2

5SPtrc

ori

lacI q

Apr

2.0

2.5

3.0

3.5

4.0 4.37/0

0.5

1.0

PvuI1.5

Nco IEcoRISst IKpnISmaIBamHIXba ISal IPstISphIHindIII

Met Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Lys Leu

ATG GAA TTC GAG CTC GGT ACC CGG GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAG CTT GGCTGTTTTGGC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

rrnB

TCACACAGGAAACAGACC

RBS

pTrc99A

Nco l EcoRI Sst I KpnI XmaISmaI

BamHI XbaI SaI IAcc IHincII

Pst I SphI HindIII

Met Gly Ile Arg AIa Arg Tyr Pro Gly IIe Leu

ATG GGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG A GTCGACCTGCAGGCATGCAAGCTTGGCTGTTTTGGC

1 2 3 4 5 6 7 8 9 10 11

rrnB

TCACACAGGAAACAGACC

RBS

pTrc99B



Pst I Sph I HindIII

Met Gly Asn Ser Ser Ser Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser Leu

ATG GGG AAT TCG AGC TCG GTA CCC GGG GAT CCT CTA GAG TCG ACC TGC AGG CAT GCA AGC TTG GCTGTTTTGGC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

rrnB

TCACACAGGAAACAGACC

RBS

pTrc99C



Pst I SphI HindIII

21

Figure 1.5.11 Map of pTrc99A,B,C.


1.5.15


ordered restriction map by end labeling andpartial restriction digestion.

pEGFP-1 is a selectable vector for monitor-ing promoter activity in mammalian cells viafluorescence of a green fluorescent protein(GFP) derivative (Clontech; see Fig. 1.5.5;Yang et al., 1996). The vector contains a neo-mycin resistance gene downstream of the SV40early promoter for selection of stably trans-formed mammalian cells. It has a polylinkerlocated upstream of the EGFP gene, so that thefunction of promoter sequences introduced intothe polylinker can be assessed based on EGFPactivity. The EGFP gene is modified from wild-type GFP to ensure expression in mammaliancells, it has silent base mutations that corre-spond to human codon-usage preferences, andsequences flanking the coding region have beenconverted to a Kozak consensus translationinitiation signal. The vector backbone containsan f1 ori for production of ssDNA, a pUC-de-rived ori for propagation in E. coli and akanamycin resistance gene for selection in bac-teria.

A series of yeast shuttle vectors (pRS304,305, and 306) has been created to facilitatemanipulation of DNA in Saccharomyces cere-visiae (see Fig. 1.5.6; Sikorski and Hieter,1989). These vectors have a backbone derivedfrom pBLUESCRIPT into which the featuresnecessary for replication and maintenance inyeast have been introduced. The members ofthis series of plasmids differ only in the yeastselectable marker incorporated; pRS303 car-ries the HIS3 marker that complements a non-reverting his3 chromosomal mutation in spe-cific yeast strains. These plasmids contain anautonomously replicating sequence as well asa centromere sequence, CEN6, that ensuresstable maintenance in yeast cells.

pcDNA3.1 is a selectable cloning and ex-pression vector for use in mammalian cells. Thefeatures of this vector include a neomycin re-sistance gene driven by the SV40 early pro-moter (contained within the SV40 ori) andterminated by an SV40 polyadenylation signalfor selection in mammalian cells (see Fig.1.5.7). In addition, due to the inclusion of theSV40 ori, the vector can replicate as an episomein cells expressing the SV40 large T antigen.The polylinker cloning site is located down-stream of strong cytomegalovirus enhancer-promoter sequences and upstream of the bovinegrowth hormone gene termination signals forhigh-level expression of protein-coding se-quences cloned into this vector. This vector alsocontains some of the more standard features of

other plasmid vectors, including a ColE1 rep-licator for propagation in E. coli, the ampicillinresistance gene for selection in E. coli, the f1ori for production of ssDNA, and the T7 pro-moter sequence for in vitro transcription ofDNA inserted into the polylinker.

pRR54 is an example of a broad-host-rangemobilizable plasmid vector. This vector con-tains replicator and stablilization sequences de-rived from the natural RK2 broad-host-rangeplasmid (see Fig. 1.5.8; Roberts et al., 1990).oriV is the vegetative origin of replication, trfAencodes trans-acting functions necessary forreplication, and par encodes a locus that en-hances stability of the plasmid. This plasmidcan be mated into diverse gram-negative spe-cies as long as the appropriate mobilizationmachinery is provided in trans because it con-tains the origin of conjugal transfer, oriT. Theplasmid carries the β-lactamase gene, allowingfor ampicillin/carbenecillin selection of plas-mid containing bacteria.

The pTrc series of plasmid expression vec-tors facilitates regulated expression of genes inE. coli. These vectors carry the strong hybridtrp/lac promoter, the lacZ ribosome-bindingsite (RBS), the MCS of pUC18 that allowsinsertion in three reading frames, and the rrnBtranscription terminators (see polylinker se-quences given below the vector diagram in Fig.1.5.11). These vectors are equally useful forexpression of unfused proteins (resulting frominsertion in the NcoI site) or for expression offusion proteins (using one of the cloning sitesin the correct translational frame). The presenceof the lacIq allele on the plasmid ensures com-plete repression of the hybrid trp/lac promoterduring cloning and growth in any host strain(see Amann et al., 1988, for further details).

LITERATURE CITEDAmann, E., Ochs, B., and Abel, K.-J. 1988. Tightly

regulated tac promoter vectors useful for theexpression of unfused and fused proteins in Es-cherichia coli. Gene 69:301-315.

Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach,M.C., Heynecker, H.L, and Boyer, H.W. 1977.Construction of useful cloning vectors. Gene2:95-113.

Chang, A.C.Y. and Cohen, S.N. 1978. Constructionand characterization of amplifiable mulitcopyDNA cloning vehicles derived from the P15Acryptic miniplasmid. J. Bacteriol. 134:1141-1156.

Gerhart, E., Wagner, H., and Simmons, R.W. 1994.Antisense RNA control in bacteria, phages, andplasmids. Annu. Rev. Microbiol. 48:713-742.


1.5.16


Jackson, D.A., Symons, R.M., and Berg, P. 1972.Biochemical method for inserting new geneticinformation into DNA of Simian Virus 40 circu-lar DNA molecules containing lambda phagegenes and the galactose operon of Escherichiacoli. Proc. Natl. Acad. Sci. U.S.A. 69:2904-2909.

Kahn, M., Kolter, R., Thomas, C., Figurski, D.,Meyer, R., Remaut, E., and Helinski, D.R. 1979.Plasmid cloning vehicles derived from plasmidsColE1, F, R6K, RK2. Methods Enzymol. 68:268-280.

Kim, U.J., Shizuya, H., de Jong, P.J., Birren, B., andSimon, M.I. 1992. Stable propagation of cosmid-sized human DNA inserts in an F-factor basedvector. NAR 20:1083-1085.

Norrander, J., Kempe, T., and Messing, J. 1983.Construction of improved M13 vectors usingoligonucleotide-directed mutagenesis. Gene26:101-106.

Roberts, R.C., Burioni, R., and Helinski, D.R. 1990.Genetic characterization of the stabilizing func-tions of a region of broad-host-range plasmidRK2. J. Bacteriol. 172:6204-6216.

Shizuya, H., Birren, B., Kim U.J., Mancin, V.,Slepak, T., Tachiiri, Y., and Simon, M.I. 1992. Abacterial system for cloning large human DNAfragments. Proc. Natl. Acad. Sci. U.S.A.89:8794-8797.

Sikorski, R.S. and Hieter, P.I. 1989. A system ofshuttle vectors and yeast host strains designed forefficient manipulation of DNA in Saccharomy-ces cerevisiae. Genetics 122:19-27.

Stoker, N.G., Fairweather, N., and Spratt, B.G.1982. Versatile low-copy-number plasmid vec-tors for cloning in Escherichia coli. Gene18:335-341.

Sutcliffe, J.G. 1978. Complete nucleotide sequenceof the Escherichia coli plasmid pBR322. ColdSpring Harbor Symp. Quant. Biol. 43:77-90.

Uhlin, B.E., Schweickart, V., and Clark, A.J. 1983.New runaway-replication-plasmid cloning vec-tors and suppression of runaway replication bynovobiocin. Gene 22:255-265.

Wahl, G.M., Lewis, K.A., Ruiz, J.C., Rothenberg,B., Zhao, J., and Evans, G.A. 1987. Cosmidvectors for rapid genomic walking, restrictionmapping, and gene transfer. Proc. Natl. Acad.Sci. U.S.A. 84:2160-2164.

Yang, T., Cheng, L., and Kain, S.R. 1996. Optimizedcodon usage and chromophore mutations pro-vide enhanced sensitivity with the green fluores-cent protein. Nucl. Acids Res. 24:4592-4593.

Contributed by Rhonda FeinbaumMassachusetts General HospitalBoston, Massachusetts


1.5.17


UNIT 1.6Minipreps of Plasmid DNA

Although there are a large number of protocols for the isolation of small quantities ofplasmid DNA from bacterial cells (minipreps), this unit presents four procedures basedon their speed and success: the alkaline lysis prep, a modification of the alkaline lysisprep that is performed in 1.5-ml tubes or 96-well microtiter dishes, the boiling method,and a lithium-based procedure. A support protocol provides information on storingplasmid DNA.

BASICPROTOCOL

ALKALINE LYSIS MINIPREP

The alkaline lysis procedure (Birnboim and Doly, 1979, and Birnboim, 1983) is the mostcommonly used miniprep. Plasmid DNA is prepared from small amounts of manydifferent cultures (1 to 24) of plasmid-containing bacteria. Bacteria are lysed by treatmentwith a solution containing sodium dodecyl sulfate (SDS) and NaOH (SDS denaturesbacterial proteins, and NaOH denatures chromosomal and plasmid DNA). The mixtureis neutralized with potassium acetate, causing the covalently closed plasmid DNA toreanneal rapidly. Most of the chromosomal DNA and bacterial proteins precipitate—asdoes the SDS, which forms a complex with potassium—and are removed by centrifuga-tion. The reannealed plasmid DNA from the supernatant is then concentrated by ethanolprecipitation.

Materials

LB medium (UNIT 1.1) containing appropriate antibiotic (Table 1.4.1)Glucose/Tris/EDTA (GTE) solutionTE buffer (APPENDIX 2)NaOH/SDS solutionPotassium acetate solution95% and 70% ethanol10 mg/ml DNase-free RNase (optional; UNIT 3.13)1.5-ml disposable microcentrifuge tubes

1. Inoculate 5 ml sterile LB medium with a single bacterial colony. Grow to saturation(overnight).

2. Spin 1.5 ml of cells 20 sec in a microcentrifuge at maximum speed to pellet. Removethe supernatant with a Pasteur pipet.

The spins in steps 2 and 6 can be performed at 4°C or at room temperature. Longer spinsmake it difficult to resuspend cells.

3. Resuspend pellet in 100 µl GTE solution and let sit 5 min at room temperature.

Be sure cells are completely resuspended.

4. Add 200 µl NaOH/SDS solution, mix by tapping tube with finger, and place on icefor 5 min.

5. Add 150 µl potassium acetate solution and vortex at maximum speed for 2 sec to mix.Place on ice for 5 min.

Be sure mixing is complete.

6. Spin 3 min as in step 2 to pellet cell debris and chromosomal DNA.

7. Transfer supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol, and let sit 2min at room temperature to precipitate nucleic acids.

Contributed by JoAnne Engebrecht, Roger Brent, and Mustak A. KaderbhaiCurrent Protocols in Molecular Biology (1991) 1.6.1-1.6.10Copyright © 2000 by John Wiley & Sons, Inc. Supplement 15

1.6.1


8. Spin 1 min at room temperature to pellet plasmid DNA and RNA.

9. Remove supernatant, wash the pellet with 1 ml of 70% ethanol, and dry pellet undervacuum.

10. Resuspend the pellet in 30 µl TE buffer and store as in support protocol. Use 2.5 to5 µl of the resuspended DNA for a restriction digest.

Contaminating RNA may interfere with detection of DNA fragments on the agarose gel; itcan be destroyed by adding 1 �l of a 10 mg/ml RNase solution (DNase-free) to the digestionmixture.

ALTERNATEPROTOCOL

ALKALINE LYSIS IN 96-WELL MICROTITER DISHES

Escherichia coli cells that contain plasmids are grown and lysed, and the plasmid DNAis precipitated—all in the wells of 96-well microtiter dishes. This procedure makes itpossible to perform hundreds of rapid plasmid preps in a day. It is based on an unpublishedprocedure by Brian Seed of Massachusetts General Hospital.

Additional Materials

TYGPN medium (UNIT 1.1)70% ethanol, ice-coldIsopropanol

96-well microtiter plates (Dynatech PS plates or equivalent)Multichannel pipetting device (8-prong Costar; 12-prong Titer Tek)Multitube vortexerSorvall RT-6000 low-speed centrifuge, or equivalent, with microplate carrier in

H-1000B rotor

1. Add 0.3 ml sterile TYGPN medium to each well of a 96-well microtiter plate (seesketch 1.6A). Inoculate each well with a single plasmid-containing colony.

The 96-well microtiter plates used must have U-shaped bottoms. To take full advantage ofthis protocol, one should perform all pipetting steps with a multichannel pipetting device.

adding solution to96-well microtiter dish

Sketch 1.6A


1.6.2

Minipreps ofPlasmid DNA

2. Grow bacteria to saturation at 37°C (∼48 hr).

All subsequent steps are performed at room temperature unless otherwise noted.

Potassium nitrate in the TYGPN medium presumably acts as a terminal electron acceptorwhen the bacteria in the wells are growing anaerobically, resulting in high cell densities.

3. Spin saturated cultures in H-1000B rotor with microplate carrier for 10 min at 2000rpm (600 × g), 4°C. Decant supernatant with brief flick.

A microplate carrier is available for the Beckman JS-4.2 rotor. The same rpm values canbe used for the JS-4.2 as those given here for the Sorvall H-1000B (see APPENDIX 1 for rotorconversion values).

4. Resuspend cells in the well bottoms by clamping the plate in a multitube vortexerand running it 20 sec at setting 4.

5. Add 50 µl GTE solution to each well.

6. Add 100 µl NaOH/SDS solution to each well. Wait 2 min.

7. Add 50 µl potassium acetate solution to each well.

8. Cover with plate tape or parafilm. Agitate vigorously in vortexer 20 sec at setting 4.Spin 5 min at 2000 rpm (600 × g), 4°C.

9. Insert a pipet tip just at the edge of the U in the bottom of the well. Remove 200 µlfrom each well and transfer to a new plate.

Do not try to recover all the fluid in each well.

10. Add 150 µl isopropanol to each well of the new plate. Cover with plate tape, agitate,and chill 30 min at −20°C.

11. Spin 25 min at 2000 rpm, 4°C, and decant supernatant. Wash pellets with cold 70%ethanol, gently decant supernatant, wash with 95% ethanol, and again gently decantsupernatant.

Pellets often shrink visibly during the 70% ethanol wash, as impurities in them aredissolved.

Restriction enzymes will not cut well if the DNA is contaminated with even very smallamounts of NaOH/SDS or potassium acetate solutions. It is therefore very important todecant the supernatants from the isopropanol precipitation and ethanol washes thoroughly,but not so vigorously that the pellets are flung out of the well bottoms.

If pellets become detached during either washing step, the plate should be respun at 2500rpm for 5 min to bring the pellets to the bottom of the wells again.

12. Air dry pellets for 30 min, then resuspend in 50 µl TE buffer. Store as in supportprotocol and use 10-µl aliquots for digestion.

Current Protocols in Molecular Biology Supplement 15 CPMB

1.6.3


BASICPROTOCOL

BOILING MINIPREP

Bacteria that contain plasmid DNA are broken open by treatment with lysozyme, Triton(a nonionic detergent), and heat. The chromosomal DNA remains attached to the bacterialmembrane and is pelleted to the bottom of a centrifuge tube during a brief spin. PlasmidDNA is precipitated from the supernatant with isopropanol (Holmes and Quigley, 1981).This procedure is recommended for preparing small amounts of plasmid DNA from 1 to24 cultures. It is extremely quick, but the quality of DNA produced is lower than that fromthe alkaline lysis miniprep.

Materials

LB medium (UNIT 1.1) containing appropriate antibiotic (Table 1.4.1)STET solutionHen egg white lysozymeIsopropanol, ice-coldTE buffer (APPENDIX 2)10 mg/ml DNase-free RNase (optional; UNIT 3.13)

1.5-ml disposable microcentrifuge tubesBoiling water bath (100°C)

1. Inoculate 5 ml sterile LB medium with a single bacterial colony. Grow at 37°C atleast until mid-log phase ∼6 hr, (a freshly saturated overnight culture works evenbetter; see UNIT 1.2).

2. Transfer 1.5 ml of the saturated culture to a 1.5-ml microcentrifuge tube and pelletthe cells by spinning 20 sec in microcentrifuge at maximum speed. Discard super-natant with a Pasteur pipet.

The spins in steps 2, 6, and 7 can be performed at 4°C or room temperature. Longer spinsmake it difficult to resuspend cells.

3. Resuspend the bacteria in 300 µl of STET solution containing 200 µg lysozyme.Vortex to achieve complete suspension.

Be sure cells are completely resuspended in order to maximize the number of cells exposedto the lysozyme and consequently the yield of plasmid DNA.

4. Place tube on ice for 30 sec to 10 min.

The time required for this step can vary between the limits indicated without affecting theyield or quality of the plasmid DNA.

5. Place tube in a boiling water bath (100°C) 1 to 2 min.

Heat and detergents cause the weakened cell walls to break, releasing plasmid DNA andRNA, but not the larger bacterial chromosome which remains attached to or trapped insidethe lysed cells.

6. Spin in microcentrifuge 15 to 30 min at maximum speed.

The pellet, which should be fairly gummy, contains bacterial debris as well as chromosomalDNA. The supernatant contains plasmid DNA and RNA.

7. Pipet off supernatant into a new tube, carefully, without dislodging pellet. Mix with200 µl (an equal volume) of cold isopropanol. Place at −20°C for 15 to 30 min. Spin5 min in microcentrifuge at maximum speed.

The cold isopropanol precipitates the plasmid DNA and cellular RNA. Considerablyshorter incubation periods (e.g., 2 to 5 min) may be sufficient for precipitation.

8. Remove the supernatant by inverting the tube and flicking it several times. Dry the


1.6.4


pellet by placing under a vacuum until it looks flaky.

If a vacuum source is unavailable, the pellet can be air dried.

9. Resuspend the pellet in 50 µl TE buffer and store as in the support protocol. Use 5µl of the resuspended DNA for a restriction digest.


BASICPROTOCOL

LITHIUM MINIPREP

Plasmid DNA is obtained from E. coli grown on plates as colonies or in liquid cultures.Bacterial cells harboring plasmid DNA are sequentially treated with Triton X-100/LiCland phenol/chloroform. These steps solubilize plasmid DNA while precipitating chromo-somal DNA with cellular debris. The debris is removed by centrifugation. This isolationprocedure yields preparations of plasmid DNA that are virtually devoid of chromosomalDNA.

The procedure described here (originally presented by He et al., 1990) for small-scaleisolation of plasmid DNA can also be readily extended for large-scale preparations asdescribed in the annotation to the final step. The merit of the approach is that it is extremelyreliable and rapid—requiring no more than 20 min of simple and economical operationsfor a preparation. The final plasmid DNA preparations are of a purity and quality usablefor most biological applications.

Materials

TELT solutionLB medium (UNIT 1.1) containing appropriate antibiotic (Table 1.4.1)l:l (w/v) phenol/chloroform (UNIT 2.1)100% ethanol, prechilled to −20°CTE buffer (APPENDIX 2)10 mg/ml DNase-free RNase A (optional; UNIT 3.13)1.5-ml disposable microcentrifuge tubes

NOTE: All steps are performed at room temperature.

1. To isolate plasmid DNA from transformant colonies grown on agar plates, preparethe cells as follows:

a. Using a microspatula, scoop out an entire bacterial colony grown to 2- to 5-mmdiameter on an LB agar plate. Transfer the colony to a 1.5-ml microcentrifuge tubecontaining 100 µl TELT solution.

b. Vortex thoroughly to suspend the cells. Proceed to step 3.

2. To isolate plasmid DNA from liquid cultures, prepare the cells as follows:

a. Inoculate a colony of bacteria into 1.8 ml of sterile LB medium supplemented withappropriate antibiotic. Grow to saturation with shaking for 18 to 24 hr at 30°C(see UNIT 1.2).Glass test tubes with plastic caps are suitable. Place the tubes at a suitably inclined angleto achieve good agitation.

b. Carefully transfer the entire culture volume into a 1.5-ml microcentrifuge tube.At this stage the volume of the liquid culture will have been reduced to ∼1.5 ml.


1.6.5


c. Pellet the cells by spinning in a microcentrifuge (10,000 × g) for 20 sec.The spins in steps 2, 4, 7, and 9 can be performed at 4°C or room temperature. Centrifu-gation for longer periods or at higher speeds makes it difficult to resuspend the cells in thefollowing step.

d. With the tube held in a vertical position, aspirate the supernatant using a long-tipped Pasteur pipet connected to a vacuum line.

e. Add 100 µl of TELT solution to the pellet and resuspend by vortexing.Ensure that the cells are thoroughly suspended. See annotation to step 11 for scaled-upDNA preparations.

3. Add 100 µl of 1:1 phenol/chloroform and thoroughly vortex for 5 sec.

This mixture may be left at room temperature for ≤15 min. Plasmid yield will elevate withincreasing duration of incubation; however, incubation periods >15 min may result inphenol-mediated modification of DNA.

4. Microcentrifuge 1 min at 15,000 × g (maximum speed).

5. Using a pipettor or similar device, carefully withdraw 75 µl of the upper aqueousphase and transfer the contents into a clean microcentrifuge tube.

Do not agitate the resolved phases. If mixing occurs, recentrifuge. When collecting the toplayer avoid picking the debris at the interface.

6. To the supernatant, add 150 µl of chilled 100% ethanol. Mix the contents well toprecipitate the plasmid DNA.

7. Pellet the nucleic acids by microcentrifuging 5 min at maximum speed.

8. Discard the supernatant by inverting the tube. When all the supernatant has drained,hold the tube in the same position for a few seconds and wipe off the last droplet fromthe rim of the tube by touching the edge of a Kleenex paper tissue.

9. Wash the pellet with 1 ml of cold 100% ethanol and harvest the nucleic acid pellet asin steps 6 and 7.

After centrifugation, decant the supernatant carefully as the pellet may be loose.

10. Cap the tube. Stab a small hole in the cap with a thumbtack or a syringe needle. Placethe tube in a vacuum desiccator (without desiccant). Apply vacuum until the nucleicacid pellet appears completely dry.

A water-pumped vacuum line suffices for the purpose and usually takes ≤15 min.

11. Dissolve the pellet in 30 µl TE buffer. Vortex the contents well, capturing most of theDNA around the inner surface of the microcentrifuge tube. Store as in the supportprotocol and use 2 to 5 µl of DNA solution in a final 20-µl reaction volume forrestriction digestion.


For scaled-up plasmid DNA preparations (He et al., 1991), increase the amounts of TELTsolution and 1:1 phenol/chloroform in direct proportion to the culture volume used. Forcultures ≤5 ml, transfer the cells after suspension in TELT buffer into a microcentrifugetube. Wash the final nucleic acids pellet twice with 1 ml of 100% ethanol. For culturesbetween 5 and 100 ml, use Corex glass tubes for treatment with TELT and phenol/chloro-form and for centrifugations (Sorvall RC-5C centrifuge at 6000 × g).


1.6.6


SUPPORTPROTOCOL

STORAGE OF PLASMID DNA

Plasmids can be maintained for a short period (up to 1 month) in bacterial strains simplyby growing on selective plates and storing at 4°C. For permanent storage, bacteriaharboring the plasmid should be grown to saturation in the presence of the appropriateselective agent. An equal volume (∼1 to 2 ml) of bacteria should be added to sterile 100%glycerol or a DMSO-based solution (recipe in UNIT 1.3) and frozen at −70°C in sterile vials.Cells taken from storage should again be grown on a selective plate (UNIT 1.1), and theplasmid DNA should be checked by restriction analysis (UNIT 3.1).

Plasmid DNA can be stored in TE buffer at 4°C for several weeks or preserved for severalyears by storing at −20° or −70°C. Most investigators prefer to store plasmids as frozenDNA, due to the widely held belief that plasmids stored in bacteria are sometimes lost,are rearranged, or accumulate insertion sequences and transposons during storage or onrevival. Although such rearrangements certainly occur during storage of plasmids inbacteria in stab vials, we are unaware of any report of rearrangements affecting plasmidsstored in frozen cells.


Glucose/Tris/EDTA (GTE) solution50 mM glucose25 mM Tris⋅Cl, pH 8.010 mM EDTAAutoclave and store at 4°C

NaOH/SDS solution0.2 N NaOH1% (wt/vol) sodium dodecyl sulfate (SDS)Prepare immediately before use

5 M potassium acetate solution, pH 4.829.5 ml glacial acetic acidKOH pellets to pH 4.8 (several)H2O to 100 mlStore at room temperature (do not autoclave)

STET solution8% (wt/vol) sucrose5% (wt/vol) Triton X-10050 mM EDTA50 mM Tris⋅Cl, pH 8.0Filter sterilize and store at 4°C

TELT solution2.5 M LiCl50 mM Tris⋅Cl, pH 8.062.5 mM Na2 EDTA4% (wt/vol) Triton X-100Store as 1- to 5-ml aliquots frozen at −20°C (do not filter sterilize or autoclave)


1.6.7


COMMENTARY

Background InformationIsolation of small quantities of plasmid

DNA from bacterial cells is essential for theanalysis of recombinant clones (UNITS 3.2 & 6.3).A myriad of plasmid DNA miniprep methodsnow exist and investigators are generally re-markably loyal to his or her own particularprotocol. This unit presents three of the mostwidely used and reliable methods—alkalinelysis, the boiling method, and a lithium-basedminiprep. The success of any of these proce-dures is largely a function of the expertise ofthe investigator; choice of method is thereforedetermined by personal preference as well asthe size and type of the plasmid and the hoststrain of E. coli. With practice, all three proto-cols yield plasmid DNA of sufficient quantityand quality for use in most enzymatic manipu-lations (Chapters 3 & 7), and in most bacterial(UNIT 1.8) and yeast (UNIT 13.7) transformationprocedures.

The procedures presented in this unit allowfor the preferential recovery of circular plasmidDNA over linear chromosomal DNA. Treat-ments with either base or detergent (used in allthree procedures) disrupt base pairing andcause the linear chromosomal DNA to denatureand separate. In contrast, because of its super-coiled configuration, covalently closed circularplasmid DNA is unable to separate and readilyreforms a correctly paired superhelical struc-ture under renaturing conditions.

In the alkaline lysis miniprep, treatmentwith SDS and NaOH breaks open bacterialcells. Subsequent addition of potassium acetatepreferentially reanneals covalently closed plas-mid DNA, while chromosomal DNA and pro-teins are trapped in a complex formed betweenthe potassium and SDS. The lysis treatment inthe boiling miniprep causes chromosomalDNA to remain attached to the bacterial mem-brane, while plasmid DNA remains in the su-pernatant. In the lithium method, treatment ofbacterial cells with Triton X-100/LiCl resultsin dissolution of the inner bacterial plasmamembrane. However, this treatment has no ef-fect on the overall morphology of the cells asobserved microscopically, nor does it lead torelease of plasmid DNA from the cells. Sub-sequent addition of phenol/chloroform leads todenaturation and precipitation of intracellularproteins. The concomitant rapid shrinkage ofthe cells preferentially expels soluble, super-

coiled plasmid DNA into the medium whileretaining chromosomal DNA and denaturedcellular protein with the bulk of the cell mass.Bacterial morphology, particularly of the cellwall envelope, is preserved under these condi-tions for at least 30 min, at which point cell lysisensues. In all preparations, chromosomal DNAis removed with cellular debris by centrifuga-tion, and soluble, supercoiled plasmid DNA isconcentrated by ethanol precipitation.

In general, all three methods provide plas-mid DNA of comparable yield and quality suit-able for most biological applications. Yield ofDNA is determined more by the type of plasmidthan by method of isolation. Plasmids derivedfrom pBR322 generally give lower (but, formost applications, sufficient) yields comparedwith the more recently derived pUC-like vec-tors, which contain a lesion in the plasmid-en-coded rop gene, causing the plasmid to bemaintained in high copy number in the cell (seeUNIT 1.5).

All three methods are successful for theisolation of small (<10-kb) plasmids. Largerplasmids are generally poorly isolated usingthe lithium method, with yields <10% ofthose in the pUC-size range. These larger-sized plasmids may be retained along withchromosomal DNA in the mesh-like structureof the cell wall. In rare instances this problemmay extend to plasmids that exist in low copynumber and to those contained in strains withunusual cell wall compositions (see discus-sion of strain considerations below). The al-kaline prep is better suited for efficient iso-lation of large-sized or low-copy-numberplasmids (see critical parameters).

Strain background is also a considerationwhen selecting a miniprep procedure. Whenlysed with detergent or heat (as in the boilingor lithium procedures), strains such as HB101and its derivatives may release a large amountof carbohydrate that contaminates plasmidDNA and inhibits many restriction endonu-cleases. In addition, HB101-related strains ex-press endonuclease A which, if not inactivated,may degrade plasmid DNA in the presence ofmagnesium (during restriction enzyme diges-tion). Plasmids harbored in these strains maybe better isolated using the alkaline lysismethod.

In general, the alkaline lysis miniprep canbe used for a variety of plasmid types and


1.6.8


sizes carried in most host strains. The boilingmethod is perhaps the most forgiving of thethree, and the lithium miniprep, although re-stricted for use with small plasmids in certainhosts, is the most rapid method.

Critical Parametersand Troubleshooting

The successful isolation of small quantitiesof DNA from bacterial cells, regardless of theprotocol, is largely dependent on the strain ofE. coli used. For example, DNA isolated fromstrains C600, DH1 and LE392 is of good qual-ity, while DNA isolated from strains HB101and the JM100 series is of lesser quality (seeTable 1.4.5). The latter strains have high nucle-ase activity (endonuclease A, which is not com-pletely inactivated by boiling), necessitatingfurther purification with a phenol extraction(UNIT 2.1) or an additional precipitation withammonium acetate (UNIT 2.1). If one protocolfails to yield DNA with a particular strain of E.coli, it would be best to try an alternativemethod. The alkaline lysis miniprep seems tobe the most consistent regardless of strain used,while the lithium method incorporates a phenolextraction and may be well suited for isolationof plasmid from strains with high nucleaseactivity.

Failure to isolate DNA using any of theprotocols could be due to DNase contaminationof the RNase. This problem can be circum-vented by leaving out or using considerably lessRNase. Other procedure-specific guidelinesare described below.

Alkaline lysis miniprep. The DNA yield cangenerally be increased by adding 500 mg/ml oflysozyme to the 100-µl suspension of cells inthe glucose/Tris/EDTA (GTE) solution (step3).

With a few variations, this protocol can alsobe used to isolate low-copy-number plasmids(UNIT 1.5), as follows: Increase the number ofstarting cells to 3 ml and include lysozyme inthe GTE solution as described above. Becauseyields are lower with strains harboring low-copy-number plasmids, it will be necessary touse at least 5 µl of DNA for a restriction digest.

If the isolated DNA fails to cut with restric-tion endonucleases, the most common cause isinadequate washing of the pellets after the etha-nol precipitation step. Precipitating the DNA asecond time with ethanol, or washing the pelletsfrom the first precipitation with 70% ethanol,will usually clean up the DNA enough forrestriction enzyme cutting.

Boiling miniprep. Failure to isolate DNA is

sometimes caused by incomplete cell lysis. Ifthis appears to be the case, try using a new bottleof lysozyme powder. Apparent failure to re-cover DNA is also sometimes caused by DNasecontamination of the freshly prepared plasmidDNA, or of the RNase. If miniprep DNA iscontaminated with DNase, it can usually becleaned up by an additional precipitation withammonium acetate (UNIT 2.1) or by phenol ex-traction (UNIT 2.1). This method should not beused with endonuclease A–containing strains(such as HB101).

Lithium miniprep. This procedure elimi-nates many of the lengthy fractionation stepscommonly employed in other minipreps ofplasmid DNA and is quite effective for isolatingplasmids ≤10 kbp. The high quality of theplasmid DNA obtained by this method has beenverified by the fact that no problems are en-countered with regard to inhibition of somesensitive restriction enzyme activities—for in-stance NdeI, which is sensitive to trace quanti-ties of impurities (see Fig. 1.6.1). Any DNAcleavage inhibition is likely to be due to con-tamination of the DNA with cellular debrisfrom the phenol/chloroform interface. How-ever, this can be readily overcome by reducingthe volume of DNA in the reaction mixture. Theplasmid DNA is suitable for direct transforma-tion of bacteria. If speed is required, DNArecovered from the aqueous phase followingphenol/chloroform extraction (after step 4) canbe used for transformation. However, suchDNA should be used immediately to minimizethe likelihood of phenol-mediated modifica-tion of DNA.

This technique has been used to purify shut-tle vectors from yeast for high-frequency back-transformation in E. coli (Ward, 1990).

Anticipated ResultsTwo to five micrograms of DNA are ob-

tained from 1.5 ml of a culture of cells contain-ing a pBR322-derived plasmid by using eitherthe alkaline lysis miniprep, the boiling proto-col, or the lithium method. Three- to five-foldhigher yields can be expected from pUC-de-rived plasmids. DNA yield from alkaline lysisin 96-well microtiter dishes (0.3 ml of cul-ture/well) is ∼2 µg for high-copy-number plas-mids (e.g., pUC plasmids). All three methodsprovide plasmid DNA of comparable quality,suitable for use in many applications.

Time ConsiderationsUsing the alkaline lysis procedure, it is pos-

sible, with practice, to produce twelve samples


1.6.9


of DNA from saturated bacterial cultures in <1hr. Although it is possible to do a large numberof samples in a single day, we recommend usingthe alkaline lysis in 96-well microtiter dishesfor such mass screenings. With the latter pro-tocol, it is easy to process two plates in 4 hr.However, an incubation period of 48 hr is oftenrequired for cells to grow to saturation in thewells of the dish.

Starting with saturated cultures, the boilingmethod allows for the isolation of twelve DNAsamples in 1 hr.

The lithium method is extremely quick—DNA samples can be processed in 20 min. Withproper organization and availability of the ma-terials, twelve plasmid preparations can be ac-complished in substantially less than 1 hr. Theentire operation involving plasmid preparation,restriction digestion, and agarose gel electro-phoresis can be easily completed within half aday.

Literature CitedBirnboim, H.C. 1983. A rapid alkaline extraction

method for the isolation of plasmid DNA. Meth.Enzymol. 100:243-255.

Birnboim, H.C. and Doly, J. 1979. A rapid alkalineextraction procedure for screening recombinantplasmid DNA. Nucl. Acids Res. 7:1513-1523.

He, M., Kaderbhai, M.A. Adcock, I., and Austen,B.M. 1991. An improved and rapid procedure forisolating RNA-free Escherichia coli plasmidDNA. Gene Anal. Tech. 8:107-110.

He, M., Wilde, A., and Kaderbhai. 1990. A simplesingle-step procedure for small-scale prepara-tion of Escherichia coli plasmids. Nucl. AcidsRes. 18:1660.

Holmes, D.S. and Quigley, M. 1981. A rapid boilingmethod for the preparation of bacterial plasmids.Anal. Biochem. 114:193-197.

Ward, A.C. 1990. Single-step purification of shuttlevectors from yeast for high frequency back-transformation into E. coli. Nucl.Acids Res.18:5319.

Contributed by JoAnne EngebrechtState University of New YorkStony Brook, New York

Roger BrentMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts

Mustak A. Kaderbhai (lithium miniprep)University College of WalesPenglais, Aberystwyth, United Kingdom

1U

1H-N

1H-S

2U

2H-N

2H-S

3U

3

H-N

3

H-S

λ

Figure 1.6.1 Restriction mapping of three preparations of plasmid DNA (pVS-WH) from transfor-mant of E. coli TB strain. The three clones are numbered 1, 2, and 3 and were treated as follows:U, uncleaved DNA; H-N, plasmid DNA doubly cut with HindIII and NdeI; H-S, plasmid DNA doublycut with HindIII and SphI; λ, HindIII-digested λ DNA. A volume of 2.5 µl of plasmid DNA (about 1µg) was used for digestion. The samples were separated on a 0.7% agarose gel containing 80 mMTris-phosphate (pH 8.0) and 1 mM EDTA using Pharmacia Submarine electrophoresis tank with acurrent of 70 mA for 1 hr. All of the three clones are positive.


1.6.10


UNIT 1.7Large-Scale Preparation of Plasmid DNA

Although the need for large quantities of plasmid DNA has diminished as techniques formanipulating small quantities of DNA have improved, occasionally large amounts ofhigh-quality plasmid DNA are desired. This unit describes the preparation of milligramquantities of highly purified plasmid DNA. The first part of the unit describes threemethods for preparing crude lysates enriched in plasmid DNA from bacterial cells grownin liquid culture. The second part describes three methods for purifying plasmid DNA insuch lysates away from contaminating RNA and protein.

Methods for crude lysate preparation—alkaline lysis (see Basic Protocol 1), boiling (seeAlternate Protocol 1), and Triton lysis (see Alternate Protocol 2)—separate chromosomaland plasmid DNA by exploiting the structural differences between these molecules.Plasmids are covalently closed and smaller than chromosomal DNA. When the cell lysateis centrifuged to pellet chromosomal DNA and cellular debris, these differences permitplasmid DNA to remain in the supernatant (see Key References for the theory behind eachprotocol). All three protocols yield a solution greatly enriched for plasmid DNA but thatstill contains significant amounts of RNA and protein. These contaminants must beremoved if certain procedures, including 5′ end labeling with T4 polynucleotide kinaseand transfection of higher eukaryotic cells, are to be performed with the DNA. Accord-ingly, three procedures are described for purifying plasmid DNA from crude lysates.CsCl/ethidium bromide density gradient centrifugation (see Basic Protocol 2) permitsseparation by the different capacities of covalently closed plasmid DNA and chromosomalDNA to bind the intercalating agent ethidium bromide. Because binding of ethidiumbromide lowers the density of DNA and plasmid DNA can bind less ethidium bromidethan chromosomal DNA, plasmid DNA forms bands in a region of greater density (lowerin the tube) than chromosomal DNA (see Fig. 1.7.1). Polyethylene glycol (PEG) precipi-tation (see Alternate Protocol 3) takes advantage of the inverse relationship betweenmacromolecular size and concentration of PEG required for precipitation. There are twochromatographic methods for purifying plasmid DNA (see Alternate Protocol 4): anion-exchange chromatography, which exploits the strong negative charge of nucleic acids,and size-exclusion chromatography, which takes advantage of the large size of plasmidDNA molecules relative to other molecules present in the crude lysate.

BASICPROTOCOL 1

PREPARATION OF CRUDE LYSATE BY ALKALINE LYSIS

Alkaline lysis is probably the most generally useful plasmid preparation procedure. It isfairly rapid, very reliable, and yields reasonably clean crude DNA that can be furtherpurified by any of the purification methods described in this unit. Plasmid-bearing E. colicells are lysed with lysozyme. The lysate is treated with NaOH/SDS solution andpotassium acetate and centrifuged to separate plasmid DNA from proteins and chromo-somal DNA. The supernatant is treated with isopropanol to precipitate plasmid DNA.

Materials

LB medium or enriched medium (e.g., superbroth or terrific broth; UNIT 1.1)containing ampicillin or other appropriate selective agent (Table 1.4.1)

Plasmid-bearing E. coli strainGlucose/Tris/EDTA solution (UNIT 1.6)25 mg/ml hen egg white lysozyme in glucose/Tris/EDTA solution (prepare fresh)0.2 M NaOH/1% (w/v) SDS [prepare fresh from 10 M NaOH and 10% (w/v)

SDS stocks]3 M potassium acetate solution, pH ∼5.5 (see recipe)

Supplement 41

Contributed by J.S. Heilig, Karen L. Elbing, and Roger BrentCurrent Protocols in Molecular Biology (1998) 1.7.1-1.7.16Copyright © 1998 by John Wiley & Sons, Inc.

1.7.1


Isopropanol70% ethanol

Sorvall GSA, GS-3, or Beckman JA-10 rotor (or equivalent)High-speed centrifuge tubes with ≥20-ml capacity (e.g., Oak Ridge centrifuge

tubes)Sorvall SS-34 or Beckman JA-17 rotor (or equivalent)

Grow and concentrate cells1. Inoculate 5 ml LB medium or enriched medium containing selective agent (most

commonly ampicillin) with a single colony of E. coli containing the desired plasmid.Grow at 37°C with vigorous shaking overnight.

2. Inoculate 500 ml LB medium or enriched medium containing selective agent in a2-liter flask with ∼1 ml of overnight culture. Grow at 37°C until culture is saturated(OD600 ≅ 4).

To increase yields, maximize aeration using a flask with high surface area (whose volumeexceeds the culture volume—i.e., is >2 liters), add baffles, and shake at >400 rpm.Alternatively, treat cultures of cells growing logarithmically with chloramphenicol toamplify the plasmids (see UNIT 1.5). Growing the bacteria in medium that supports highercell densities also increases the yield. These media include M9, terrific broth, and LBmedium containing 0.1% (w/v) glucose (UNIT 1.1). These media can increase plasmid yields2- to 10-fold; different plasmids respond to the media differently. Most plasmids commonlyused today, particularly derivatives of the pUC series (Fig. 1.5.2), grow at a copy numberhigh enough to routinely yield 1 to 5 mg plasmid DNA from a 500-ml culture grown in LBmedium.

An important consideration when using enriched medium is the method to be used for finalpurification of plasmid DNA. Increased yield poses no problems when using CsCl/ethidiumbromide or PEG purification. In addition, commercially available chromatography col-umns have been designed for large-scale plasmid purification. For example, the largerQiagen-tip columns can purify 500 �g to 10 mg of plasmid DNA.

3. Collect cells by centrifuging 10 min at 6000 × g (∼6000 rpm in Sorvall GSA/GS-3or Beckman JA-10 rotors), 4°C.

If necessary the pellets can be stored frozen indefinitely at −20° or −70°C.

4. Resuspend pellet from 500-ml culture in 4 ml glucose/Tris/EDTA solution andtransfer to high-speed centrifuge tube with ≥20-ml capacity.

Lyse the cells5. Add 1 ml of 25 mg/ml hen egg white lysozyme in glucose/Tris/EDTA solution.

Resuspend the pellet completely in this solution and allow it to stand 10 min at roomtemperature.

Neither glucose nor lysozyme is absolutely necessary for the success of the procedure.Glucose serves as a buffer in step 6 when the pH of the solution is greatly increased byaddition of NaOH. Glucose provides buffering in the range of pH 12 and, by preventingthe pH from rising too drastically in step 6, increases the efficiency of precipitation in step7 (when the pH is lowered by addition of potassium acetate).

Lysozyme assists in the destruction of bacterial cell walls and subsequent release of plasmidDNA. Bacterial debris and soluble proteins are precipitated in step 7. One problem thatcan reduce recovery of plasmid DNA is inefficient separation of plasmid DNA from cellulardebris. Lysozyme helps increase yield by reducing the amount of plasmid DNA trapped inpartially degraded cell material and subsequently lost by precipitation at step 7. Omittinglysozyme reduces plasmid recovery by 5% to 10%.


1.7.2

Large-ScalePreparation ofPlasmid DNA

The effort and expense required to include glucose and lysozyme in step 5 is negligible. Theefficiency gained in streamlining the procedure by omitting them is also negligible.However, the potential for loss of plasmid DNA when these components are not includedis measurable and worth avoiding. It should be noted that some commercially availablechromatographic systems (e.g., Qiagen) do not use lysozyme for lysate preparation. TheQiagen modified alkaline lysis procedure relies on inefficient bacterial lysis (facilitated bysodium hydroxide lysis and acidic potassium acetate neutralization) to reduce contamina-tion of plasmid DNA with chromosomal DNA. Although omitting lysozyme reduces therecovery of plasmid DNA, the Qiagen protocol yields high-quality DNA. When usingQiagen-tips for large-scale plasmid preparation, the manufacturer’s recommendationsshould be followed.

When chromatographic methods are used for final purification of plasmid DNA, it isessential to degrade RNA that contaminates the lysate and will copurify with plasmid DNA.Treating the lysate with RNase A is the most efficient and economical method for degradingRNA. This can be accomplished at any step in the preparation of crude lysate, but it is mostconvenient to do it at step 5, by adding RNase A to the glucose/Tris/EDTA solution to afinal concentration of 50 �g/ml.

6. Add 10 ml freshly prepared 0.2 M NaOH/1% SDS and mix by stirring gently with apipet until solution becomes homogeneous and clears. Let stand 10 min on ice.

The solution should become very viscous.

7. Add 7.5 ml of 3 M potassium acetate solution and again stir gently with a pipet untilviscosity is reduced and a large precipitate forms. Let stand 10 min on ice.

8. Centrifuge 10 min at 20,000 × g (13,000 rpm in Sorvall SS-34; 12,500 rpm inBeckman JA-17), 4°C.

A large, fairly compact pellet will form; this contains most of the chromosomal DNA,SDS-protein complexes, and other cellular debris. Plasmid DNA remains in the translucentsupernatant.

Addition of ∼0.5 ml chloroform before the centrifugation can help reduce floating material.

Precipitate plasmid DNA9. Decant the supernatant into a clean centrifuge tube. Pour it through several layers of

cheesecloth if any floating material is visible. Add 0.6 vol isopropanol, mix byinversion, and let stand 5 to 10 min at room temperature.

If the supernatant is cloudy or contains floating material, repeat centrifugation (step 8)before adding isopropanol.

10. Recover nucleic acids by centrifuging 10 min at 15,000 × g (11,500 rpm in SS-34rotor; 10,500 rpm in JA-17 rotor), room temperature.

11. Wash the pellet with 2 ml of 70% ethanol; centrifuge briefly at 15,000 × g, roomtemperature, to collect pellet. Aspirate ethanol and dry pellet under vacuum.

The pellet can be stored indefinitely at 4°C.


1.7.3


ALTERNATEPROTOCOL 1

PREPARATION OF CRUDE LYSATE BY THE BOILING METHOD

The boiling method is extremely simple and fast but typically yields crude DNA contain-ing more contaminating bacterial DNA and proteins than other methods. In this protocola bacterial cell lysate is boiled to denature chromosomal DNA and protein. Thesedenatured macromolecules are precipitated by centrifugation, whereas plasmid DNAremains in the supernatant. The plasmid DNA is then precipitated with isopropanol.

Additional Materials (also see Basic Protocol 1)

STET solution (UNIT 1.6)10 mg/ml hen egg white lysozyme in 25 mM Tris⋅Cl, pH 8.0 (prepare fresh)

Boiling and ice-water bathsSorvall HB-4 rotor (or equivalent) and appropriate centrifuge tube

1. Grow and concentrate cells (see Basic Protocol 1, steps 1 to 3).

2. Resuspend pellet from a 500-ml culture in 20 ml STET solution and transfer to a glasstube or flask.

3. Add 2 ml hen egg white lysozyme and mix solution by inverting several times. Heatto near boiling over an open flame, then incubate 1 min in a boiling water bath.

Be certain that enough room remains between the top of the solution and the top of the tubeto permit safe handling and to prevent boiling over. Submerge the tube deep enough intothe boiling water to allow the entire solution to heat rapidly.

4. Place tube into an ice-water bath to cool.

5. Pour solution into centrifuge tube and centrifuge 20 min at ≥25,000 × g, preferablyin a swinging-bucket rotor (e.g., 12,000 rpm in HB-4), room temperature.

After boiling, the solution will be extremely viscous due to denatured chromosomal DNA.It will tend to behave as a gooey, semisolid mass. Therefore, be careful not to allow thesolution to overflow when pouring it into centrifuge tubes.

Centrifugation in a swinging-bucket rotor permits concentration of chromosomal DNA anddenatured proteins at the bottom of the tube in a more compact pellet than is possible in afixed-angle rotor. However, fixed-angle rotors can be used—e.g, Sorvall SS-34 at 47,000 ×g (20,000 rpm), Beckman JA-17 at 40,000 × g (17,000 rpm), Beckman 70Ti at 200,000 ×g (44,000 rpm), or SW-41 at 100,000 × g (25,000 rpm).

6. Decant supernatant to a clean centrifuge tube.

The supernatant can be used without further treatment for purifying plasmid DNA byCsCl/ethidium bromide equilibrium gradient centrifugation (see Basic Protocol 2). If thevolume is greater than necessary and inconvenient to handle, plasmid DNA can beprecipitated with isopropanol. Precipitation is required for purification by PEG precipita-tion (see Alternate Protocol 3) or column chromatography (see Alternate Protocol 4).

7. Add 0.6 vol isopropanol, mix by inversion, and let stand 5 to 10 min at roomtemperature.

8. Pellet nucleic acids by centrifuging 10 min at 15,000 × g (11,500 rpm in SS-34 rotor;10,500 rpm in JA-17 rotor), room temperature.

9. Wash the pellet with 2 ml of 70% ethanol. Centrifuge briefly to collect pellet. Aspirateethanol and dry pellet under vacuum.



1.7.4


ALTERNATEPROTOCOL 2

PREPARATION OF CRUDE LYSATE BY TRITON LYSIS

The method described below is a modification of that described by Clewell and Helinski(1970, 1972) in which Brij-58 and sodium deoxycholate were used. In this protocolplasmid DNA is extracted from a bacterial cell lysate that has been treated with TritonX-100 (TX-100). This is a very gentle procedure and is therefore useful for isolating verylarge plasmids such as cosmids. Plasmid DNA can be further purified by any of themethods described in the second part of this unit.


Sucrose/Tris/EDTA solution (see recipe)10 mg/ml hen egg white lysozyme in 25 mM Tris⋅Cl, pH 8.0 (prepare fresh)0.5 M EDTA (APPENDIX 2)10 mg/ml DNase-free RNase (UNIT 3.13)Triton lysis solution (see recipe)1:1 (v/v) buffered phenol/chloroform (UNIT 2.1)24:1 (v/v) chloroform/isoamyl alcohol (UNIT 2.1)

Additional reagents and equipment for phenol/chloroform extraction (UNIT 2.1)

1. Grow and concentrate cells (see Basic Protocol 1, steps 1 to 3).

2. Resuspend pellet from a 500-ml culture in 5 ml sucrose/Tris/EDTA solution. Transferto appropriate centrifuge tube (tube should be less than ∼1⁄3 full).

3. Add to tube:

1.5 ml 10 mg/ml hen egg white lysozyme in 25 mM Tris⋅Cl2 ml 0.5 M EDTA25 µl 10 mg/ml DNase-free RNase.

Let stand 15 min on ice.

4. Overlay with 2.5 ml Triton lysis solution. Mix gently but thoroughly by inversion andlet stand 20 min at 4°C.

This solution should not be vortexed or shaken hard because that will shear chromosomalDNA and prevent it from precipitating in the next step. The solution will become extremelyviscous as the cells lyse. Streaks of opaque material will be visible and may remainthroughout the incubation.

5. Centrifuge 70 min at 40,000 × g (18,000 rpm in Sorvall SS-34; 17,000 rpm inBeckman JA-17), 4°C.

6. Decant supernatant carefully to a clean centrifuge tube.

Avoid contaminating plasmid DNA with the gelatinous pellet. The pellet contains chromo-somal DNA and cellular debris. The pellet may detach from the bottom of the tube, so it ishelpful to hold a Pasteur pipet against the mouth of the tube while pouring to prevent thepellet from sliding into the clean tube. The integrity of the pellet varies greatly betweenpreparations; occasionally it is necessary to leave behind some of the viscous supernatantto avoid contaminating it with pellet material.

This supernatant can be used without further treatment for purifying plasmid DNA byCsCl/ethidium bromide equilibrium gradient centrifugation (see Basic Protocol 2). If thevolume is greater than necessary and inconvenient to handle, plasmid DNA can beextracted and precipitated with isopropanol. Extraction and precipitation are required forpurification by PEG precipitation (see Alternate Protocol 3) or column chromatography(see Alternate Protocol 4).


1.7.5


7. Extract the supernatant with 1:1 buffered phenol/chloroform, then with 24:1 chloro-form/isoamyl alcohol. Add 0.6 vol isopropanol to the final aqueous phase and letstand 5 to 10 min at room temperature.

Phenol extraction and isopropanol precipitation are done as described in UNIT 2.1.

8. Pellet nucleic acids by centrifuging 10 min at 15,000 × g (11,500 rpm in SS-34 rotor;10,500 rpm in JA-17 rotor), room temperature.

9. Wash the pellet with 2 ml of 70% ethanol and centrifuge briefly to collect pellet.Aspirate ethanol and dry pellet under vacuum.


BASICPROTOCOL 2

PURIFICATION OF PLASMID DNA BY CsCl/ETHIDIUM BROMIDEEQUILIBRIUM CENTRIFUGATION

This purification procedure yields high-quality plasmid DNA free of most contaminants,but it requires the use of ethidium bromide (a mutagen) and often requires long ultracen-trifuge runs to establish the density gradient. A crude bacterial cell lysate is mixed withcesium chloride (CsCl) and ethidium bromide and centrifuged to equilibrium. Ethidiumbromide is removed by passing plasmid DNA over a cation exchange column, and CsClis removed by ethanol precipitation.

Materials

Pellet from crude lysate of plasmid-bearing bacterial cell culture (see BasicProtocol 1 or Alternate Protocol 1 or 2)

TE buffer, pH 7.5 (APPENDIX 2)Cesium chloride10 mg/ml ethidium bromide (APPENDIX 2)CsCl/TE solution (see recipe)Dowex AG50W-X8 cation-exchange resin (see recipe)TE buffer (pH 7.5)/0.2 M NaCl100% and 70% ethanol

Beckman VTi65 or VTi80 rotor (or equivalent)5-ml quick-seal ultracentrifuge tubes3-ml syringes with 20-G needles

Additional reagents and equipment for ethanol precipitation (UNIT 2.1)

CAUTION: Ethidium bromide is a mutagen and environmental hazard. It should behandled carefully with gloves and disposed of properly. Methods for disposal varybetween different institutions. Consult the institution’s environmental safety office for thepreferred means of storage and disposal of ethidium bromide–containing waste.

1. Resuspend pellet from a crude lysate preparation in 4 ml TE buffer. Add 4.4 g CsCl,dissolve, and add 0.4 ml of 10 mg/ml ethidium bromide. If using supernatantsresulting from boiling or Triton lysis preparations, add 1.1 g CsCl/ml supernatant and0.1 ml of 10 mg/ml ethidium bromide/ml supernatant.

Ethidium bromide will form a complex with protein remaining in the solution to form adeep red flocculent precipitate. This can be removed by centrifuging the lysate–CsCl/ethid-ium bromide solution 5 min at ∼2000 × g, room temperature. After this procedure, theprotein–ethidium bromide complex will form a disc at the top of the solution. The solutioncan be pipetted out from beneath the disc or poured carefully, allowing the floating disc toadhere to the side of the tube.


1.7.6


2. Transfer the solution to a 5-ml ultracentrifuge tube. Top up the tube, if necessary,with CsCl/TE solution and seal tube. Band plasmid by centrifuging 3.5 hr at 500,000× g (77,000 rpm in VTi80 rotor) or ≥14 hr at 350,000 × g (65,000 rpm in VTi80 rotor;58,000 rpm in VTi65 rotor), 20°C.

This centrifugation must be done at a temperature no lower than 15°C. Because of the highconcentration of CsCl and the high centrifugal force necessary to establish the gradient,lower temperatures will cause the CsCl at the bottom of the tube (where the density ishighest) to precipitate during the run. CsCl precipitate moves the center of mass towardsthe bottom of the tube. This can unbalance the rotor and cause breakage of the rotor anddestruction of the centrifuge at least, and serious personal injury at worst. Also, equilibriumis achieved more quickly by warmer gradients.

Other rotors can be used, including fixed-angle and swinging-bucket rotors (e.g., centrifuge≥24 hr at 56,000 rpm in Ti70 rotor). These rotors require longer centrifugation times thanvertical rotors, but allow larger volumes or more samples to be included in the run.

3. Carefully remove the tube from the centrifuge. Insert a 20-G needle gently into thetop of the tube. Recover the plasmid band (the lower of the two bands) by insertinga 3-ml syringe with a 20-G needle attached into the side of the tube ∼1 cm below theplasmid band as shown in Figure 1.7.1. Insert the needle with the beveled side up.

Do not allow the gradient to be mixed by rough handling or turbulence. Be certain not tocover the top of the first needle with gloved finger. The needle serves to provide an inlet forair to displace the volume of solution being withdrawn.

If chromosomal DNA has been thoroughly removed in previous steps, only the plasmid bandmay be visible. Large amounts of plasmid DNA will be visible in the gradient in ordinarylight. Smaller amounts can be visualized more easily by side illumination with low-intensityshortwave UV light. Prolonged exposure of the DNA–ethidium bromide complex to UVlight will cause damage to the DNA and should be avoided. RNA may be detected as adiffuse region of fluorescence at the bottom of the tube.

CAUTION: To avoid potentially serious eye injury by UV light, wear UV-blocking glassesor face shield. Wear gloves when handling ethidium bromide.

chromosomal DNA

plasmid DNA

Figure 1.7.1 Collecting plasmid DNA from a CsCl gradient.


1.7.7


Protein–ethidium bromide complexes will pellet on the outside edge of the tube if notremoved earlier. To prevent contamination of plasmid DNA, avoid this area when insertingthe collection needle.

There should be no resistance in the syringe when drawing off the plasmid DNA. If thereis resistance, check that needles are clear. Occasionally the needle will become clogged ifa piece of tube enters it. Do not try to draw harder on the syringe, as this may createturbulence in the tube when the obstruction is sucked in and cause mixing of the gradient.Instead, insert another needle and use it to draw off the band. Leave the clogged needle inplace in the tube. (If the clogged needle is removed, the tube will empty through the holethat remains.) The air inlet needle can also become clogged; if it does, remove it and allowair to enter through the remaining hole. If plasmid DNA is drawn through a very smallopening in a clogged needle, it may be sheared.

4. If higher-purity plasmid DNA is required, perform a second ultracentrifugation toeliminate any contaminating RNA or chromosomal DNA. Add plasmid DNA bandto another ultracentrifuge tube, top up with CsCl/TE solution containing 1.0 mg/mlethidium bromide, and repeat steps 2 and 3.

5. Pour a Dowex AG50W-X8 column, 1.5 to 2 times the volume of the plasmidDNA/ethidium bromide solution, in a glass or plastic column. Pass several volumesof TE buffer/0.2 M NaCl through the column to wash and equilibrate it.

The column can be set up in a Pasteur pipet plugged with a little glass wool or in acommercially purchased plastic column.

6. Load the plasmid DNA/ethidium bromide solution directly from syringe to top ofresin bed without disturbing the resin.

7. Begin collecting the solution flowing through the column immediately after loadingthe plasmid solution. Wash the column with a volume of TE buffer/0.2 M NaCl equalto twice that of the volume of plasmid solution loaded.

The final volume collected from the column should be three times the volume of plasmidDNA solution removed from the gradient.

As the plasmid DNA flows through the column the ethidium bromide will be retained in theresin and form a red band at the top. All of the DNA will flow through in the volumerecommended. This procedure will dilute the CsCl sufficiently to allow the DNA to beprecipitated.

Ethidium bromide can also be removed by extracting the plasmid DNA–ethidium bromidesolution with an equal volume of TE-saturated n-butanol (UNIT 2.1). Shake the tube or vortexit vigorously to maximize the efficiency of extraction. Remove the organic upper phase andextract the aqueous phase repeatedly until no red color remains. Dilute the solution 3-foldwith TE buffer to dilute the CsCl. This procedure generates contaminated organic solventwaste. Follow correct procedure for its disposal.

8. Ethanol precipitate plasmid DNA, using 2 vol of 100% ethanol at room temperatureor −20°C, and centrifuge 10 min at 10,000 × g, 4°C.

Do not cool this solution below −20°C, as this may cause the CsCl to precipitate.

An alternative to ethanol precipitation at this step is to dialyze the plasmid DNA, fromwhich the ethidium bromide has been removed, against 500 to 1000 vol TE buffer. Dialysisbuffer should be changed at least twice with ≥2 hr between changes at room temperature,or 4 hr at 4°C (see APPENDIX 3C).

9. Wash pellet with 70% ethanol and dry under vacuum. Resuspend pellet in TE bufferand store at 4°C.


1.7.8


ALTERNATEPROTOCOL 3

PLASMID DNA PURIFICATION BY PEG PRECIPITATION

Polyethylene glycol (PEG) precipitation is a rapid, reliable, and convenient method forplasmid DNA purification. It can be stopped at any step without affecting the ultimaterecovery of plasmid DNA. No ultracentrifugation is required and the use of ethidiumbromide (a mutagen) is avoided. RNA and chromosomal DNA contaminants are removedfrom a crude lysate pellet by treating it with RNase, NaOH/SDS, and potassium acetate.Plasmid DNA is extracted from the supernatant and precipitated with PEG. This methodis suitable for preparing plasmid DNA for procedures that require plasmid DNA free ofany contaminants.


Glucose/Tris/EDTA solution (UNIT 1.6)0.2 M NaOH/1% (w/v) SDS [prepare fresh from 10 M NaOH and

10% (w/v) SDS stocks]10 mg/ml DNase-free RNase (UNIT 3.13)3 M potassium acetate solution, pH ∼5.5 (see recipe)Buffered phenol (UNIT 2.1)24:1 (v/v) chloroform/isoamyl alcohol (UNIT 2.1)10 M ammonium acetate (APPENDIX 2)PEG solution (see recipe)3 M sodium acetate, pH 5.5 (APPENDIX 2)

Sorvall SS-34 or Beckman JA-17 rotor (or equivalent)Sorvall HB-4 rotor

Remove contaminants from crude lysate pellet1. Resuspend the pellet obtained in the final step of crude lysate preparation in 1 ml

glucose/Tris/EDTA solution.

2. Add RNase to a final concentration of 20 µg/ml and incubate 20 min at 37°C.

3. Add 2 ml freshly prepared 0.2 M NaOH/1% SDS, mix by inverting the tube, and letstand 5 to 10 min at room temperature.

4. Add 1.5 ml of 3 M potassium acetate solution, mix by inverting the tube, and let stand5 to 10 min at room temperature.

5. Centrifuge 10 min at 20,000 × g (11,000 rpm in SS-34 rotor; 12,500 rpm in HB-4 orJA-17 rotor), room temperature.

If restriction fragments of the plasmid are to be prepared for use as probes in filterhybridizations, gel purification will rid the desired fragments of any contaminating tracesof chromosomal DNA. In that case steps 1 to 5 can be omitted. Resuspend the pellet fromthe crude lysate in 4 ml TE buffer and add RNase to 20 �g/ml final. Incubate 20 min at37°C and treat as described in steps 7 to 12 of this protocol.

6. Transfer the supernatant to a clean tube.

The white precipitate is primarily SDS-potassium complex. It includes any chromosomalDNA that remained in the crude lysate. The amount remaining after crude lysate prepara-tion varies. It may not be necessary to remove the chromosomal DNA completely, dependingon the procedures to which the plasmid DNA is to be subjected. However, some procedures,such as hybrid selection (UNIT 6.8), require that plasmid DNA be free of any contaminants.

7. Extract plasmid DNA with buffered phenol, then with 24:1 chloroform/isoamylalcohol.


1.7.9


8. Add 1⁄4 vol of 10 M ammonium acetate (2 M final concentration) to the aqueous phaseand mix. Add 2 vol of 100% ethanol and place tube in dry ice for 10 min.

9. Recover plasmid DNA by centrifuging 10 min at 10,000 × g (8000 rpm in HB-4 orSS-34 rotor; 8500 rpm in JA-17 rotor), 4°C.

10. Wash the pellet with 70% ethanol and dry briefly under vacuum.

PEG precipitate plasmid DNA11. Resuspend the pellet in 2 ml TE buffer and add 0.8 ml PEG solution. Incubate 1 to

15 hr at 0°C.

The percentage of plasmid DNA recovered with PEG precipitation increases with time ofincubation at 0° to 4°C. Usually ≥50% (∼0.5 to 3 mg) can be recovered by centrifuging thePEG-precipitated DNA solution after 1 hr at 0°C. Incubating the supernatant ≥12 hr at4°C will permit complete recovery of remaining plasmid DNA while plasmid DNA collectedafter the first centrifugation can be used for other procedures.

12. Recover plasmid DNA by centrifuging 20 min at 10,000 × g (8000 rpm in HB-4 orSS-34 rotor; 8500 rpm in JA-17 rotor; 10,000 rpm in most microcentrifuges), 4°C.

13. Resuspend pelleted plasmid DNA in 1 ml TE buffer. Ethanol precipitate plasmidDNA using 3 M sodium acetate, pH 5.5.

ALTERNATEPROTOCOL 4

PLASMID DNA PURIFICATION BY ANION-EXCHANGE OR SIZE-EXCLUSION CHROMATOGRAPHY

Chromatographic methods for purifying plasmid DNA take advantage of distinctionsbetween the physical properties of plasmid DNA and those of molecules that copurifywith it in the crude lysate. Nucleic acids are negatively charged and can therefore bepurified away from contaminants using anion-exchange chromatography (see UNIT 2.1B fora protocol and discussion of one anion-exchange method). Similarly, the large size ofplasmid DNA allows it to be purified away from smaller contaminants by gel-filtrationchromatography. The specific properties of most matrices provided by commercialsuppliers are unknown, due to the reluctance of the suppliers to divulge proprietaryinformation, but they typically exploit one or both of these methods.

This protocol describes modifications for preparing a crude lysate for chromatographicpurification of plasmid DNA and discusses features of three commercially availablecolumns. Because no single protocol is appropriate for all of the chromatographymethods, it is important to adhere to the manufacturer’s suggested methodology. Mostcolumns are supplied as kits that include reagents for preparing crude lysate using alkalinelysis (see Basic Protocol 1; e.g., Qiagen Plasmid Kits). Most manufacturers will alsoprovide the columns separately.

Preparation of Crude Lysate

It is important to match the bacterial culture volume and cell density to the quantity ofDNA expected and the plasmid DNA binding capacity of the column to be used. Forlarge-scale plasmid preparation, most commercial suppliers provide a range of columnsizes, with plasmid DNA yields of up to 10 mg. If too much culture volume is used,alkaline lysis will be inefficient, the column will be overloaded, and the performance ofthe system reduced. Furthermore, the excessive viscosity of the lysate will requirevigorous mixing, resulting in shearing of bacterial genomic DNA and subsequent con-tamination of the plasmid DNA.


1.7.10


Successful chromatographic purification of DNA using the Qiagen-tips requires that thebacterial cells be incompletely lysed, so lysozyme should be omitted from the preparationof crude lysate.

The most frequent contaminant of plasmid DNA prepared by chromatographic methodsis high-molecular-weight RNA. This contamination is reduced by adding 50 µg/ml RNaseA (from frozen 1 mg/ml stock, UNIT 3.13) to the resuspended cell pellet. The reagentsprovided with Qiagen-tips contain RNase A. However, RNase A is stable for no more thanseveral months at 4°C, the storage temperature of the working solutions. The Qiagenprotocol uses RNase at 100 µg/ml; following addition of the RNase to the bacterial pelletresuspension buffer (P1), it is stable for 6 months at 4°C. The reagents provided by 5Prime→3 Prime include a mixture of RNase A and RNase T1. The manufacturer suggestsadding the RNases to a resuspended crude lysate pellet (see Basic Protocol 1, step 11)because the pellet is resuspended in a smaller volume at this step and less RNase isrequired.

Column flow is greatly impeded or completely prevented by the presence of solid materialin the lysate when it is loaded onto the column. The most common source of solids isfloating material from the precipitation (see Basic Protocol 1, step 7). To be certain thatno floating material is loaded onto the column, decant the supernatant through cheese-cloth, add chloroform before centrifugation, or recentrifuge the supernatant.The Qiagenprotocol allows the lysate to be loaded on the Qiagen-tip column directly followingremoval of the protein and cellular debris by precipitation and centrifugation (see BasicProtocol 1, steps 7 to 9). Alternatively, Qiagen supplies a Qiafilter which rapidly filtersbacterial lysates without centrifugation. The Qiafilter is available in syringe form forfiltration of lysates from ≤250 ml of culture, or in cartridge format for filtering lysatesfrom ≤2.5 liters of culture. These filtration units efficiently remove protein, cell debris,and even small SDS precipitates. Following lysate clearing using either centrifugation orfiltration through a Qiafilter, the plasmid DNA can be purified directly on a Qiagen-tipcolumn. The pZ523 protocol requires the crude lysate plasmid DNA to be precipitatedwith isopropanol. This reduces the volume of material to be loaded on the column andallows the buffer in which plasmid DNA is loaded to be optimized for the column.Isopropanol precipitation can be used with either of the alternate protocols for preparationof crude lysate, and the final pellet can be resuspended in the buffer appropriate for thechromatographic matrix to be used.

Column Capacity

Overloading columns with large lysate volumes can affect separation characteristics,leading to reduced yield and purity. Suppliers offer a range of column sizes to allowmaximum plasmid DNA binding and recovery from large bacterial lysate volumes. Themaximum plasmid-binding capacities of Qiagen tips 2500 and 10000 are 2.5 mg and 10mg, respectively. The pZ523 column has a capacity of 4 to 5 mg and does not require thatDNA bind to the column, so overloading is less likely to be a problem. The standardprotocols for most other commercial columns are adjusted to provide a “good” yield fora plasmid that is maintained at a moderate copy number when cells that bear it are grownin ∼500 ml LB medium.

To optimize plasmid DNA recovery, culture volume, culture medium, plasmid copynumber, size of the insert, and the host strain should be considered. Very-low-copy-num-ber plasmids and cosmids often require large culture volumes to yield significant amountsof DNA. When preparing DNA from high-copy-number plasmids such as pUC and itsderivatives, culture volumes one-fourth to one-half the standard volume may be appro-priate. When using media that support growth to high cell density such as terrific broth(UNIT 1.1; Tartoff and Hobbs, 1987), culture volumes one-fourth to one-tenth the standard


1.7.11


may be appropriate. The protocol provided by Qiagen includes a table of recommendedculture volumes for plasmid isolation from a variety of sources, including high- tovery-low-copy-number plasmids.

Plasmid DNA exceeding the capacity of the column will in no way prevent recovery ofDNA. The excess DNA will simply run through the column and be discarded. One wayto increase the yield is to recover and save the material that flows through the columnwhen it is initially loaded. Some columns can be regenerated following elution of theplasmid DNA and the initial flowthrough reloaded. Qiagen resin is stable for up to 6 hrafter equilibration and Qiagen-tips may be reused within 6 hr for the same sample. Beyondthis time the separation characteristics of the resin will begin to change. The manufac-turer’s recommendations should be followed. Columns such as pZ523, which requirevacuum and centrifugation respectively, collapse during use and cannot be reused. In thiscase, excess DNA in the flowthrough can be purified on a second column of the sametype.


Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 4.

CsCl/TE solution100 ml TE buffer, pH 7.5 (APPENDIX 2)100 g CsClStore indefinitely at room temperature

Dowex AG50W-X8 cation exchange resinPrepare large batches (200 to 400 ml packed resin) of Dowex AG50W-X8 resin (100-to 200-mesh; Bio-Rad) by performing the following series of washing steps. Use alarge Buchner funnel and filter paper to collect the resin between changes of washsolution.1. Wash resin in ≥10 vol of 0.5 N NaOH until no color is observed in wash

solution (resin will retain its buff color).2. Wash with 5 to 10 vol of 0.5 N HCl.3. Wash with 5 to 10 vol of 0.5 M NaCl.4. Wash with 5 to 10 vol of distilled H2O.5. Wash with 5 to 10 vol of 0.5 N NaOH.6. Wash with distilled H2O until pH = 9.7. Store prepared resin indefinitely in 0.5 M NaCl/0.1 M Tris (pH 7.5) at 4°C.

Polyethylene glycol (PEG) solution30% (w/v) PEG 80001.6 M NaClStore indefinitely at 4°C

Potassium acetate solution (3 M), pH ∼5.5294 g potassium acetate (3 M final)50 ml 90% formic acid (1.18 M final)H2O to 1 literStore indefinitely at room temperature

Sucrose/Tris/EDTA solution25% (w/v) sucrose50 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)100 mM EDTA, pH 8.0 (APPENDIX 2)Store indefinitely at 4°C


1.7.12


Triton lysis solution3% (v/v) Triton X-100200 mM EDTA, pH 8.0 (APPENDIX 2)150 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)Store indefinitely at 4°C

COMMENTARY

Critical Parameters andTroubleshooting

Preparation of crude lysate by alkalinelysis. This is a reliable procedure and one inwhich few irretrievable disasters can occur. Onepotential problem is failure of chromosomalDNA and proteins to precipitate after additionof potassium acetate solution. The cause of thisis probably improper pH of the potassium ace-tate solution. The NaOH/SDS solution dena-tures linear (chromosomal) DNA. When thesolution is neutralized in the presence of a highsalt concentration (as when potassium acetatesolution is added), the linear DNA precipitates.This precipitation presumably is due to inter-strand reassociation of denatured, linear DNAmolecules at multiple sites. It results in theformation of a large, insoluble DNA network.Protein–SDS complexes also precipitate underthese conditions. If the pH of the potassiumacetate solution is not ∼5.5, these precipitateswill not form. If precipitation fails to occurbecause the pH is incorrect, the preparation canbe saved by adding concentrated formic oracetic acid dropwise to the solution. Mix aftereach addition until the viscosity decreases,which will happen suddenly. A precipitate willthen appear.

Preparation of crude lysate by the boilingmethod. The drawback to this procedure is thatif it fails there are few chances for recovery. Theonly easily assayed step is the final recovery ofplasmid DNA. Failure is often caused by inac-tive lysozyme or incorrect boiling time. Anindication of failure will be that the solution isnot extremely viscous following step 4. Theremedy for inactive lysozyme is to try a newpreparation of lysozyme. The optimal boilingtime may vary slightly between bacterialstrains. Using a strain for which the correct timeis already known is the simplest remedy to thisproblem. Alternatively, the correct time may bedetermined empirically by performing the boil-ing miniprep procedure in UNIT 1.6.

Preparation of crude lysate by Triton lysis.This method is much gentler than the othersdescribed in this unit because of the relativelymild conditions used to disrupt the cells (i.e.,

use of Triton X-100 and lysozyme rather thanboiling or severe changes in ionic strength andpH). Because the conditions used are near thelower limits of their effectiveness, solutionsmust be prepared correctly and the lysozymemust be active. Although powdered lysozymeis stable for long periods when stored properly,it is occasionally necessary to purchase and usea new bottle. The solution should become ex-tremely viscous when Triton lysis solution isadded, indicating that lysis has occurred. Cen-trifugation is also critical for separating plas-mid DNA from the bulk of contaminants. If thepellet is not reasonably firm at this stage it willpour out of the tube when the plasmid DNA–containing supernatant is decanted. If this hap-pens, repeat the centrifugation. If it happensroutinely, increase the reagent volumes propor-tionately throughout the procedure or centri-fuge for longer times or at higher g forces, ifnecessary using an ultracentrifuge.

Plasmid DNA purification byCsCl/ethidium bromide centrifugation. It isimportant that the density of the DNA–CsCl/ethidium bromide solution be correct forthis procedure to work. Therefore, be precise(using graduated plastic tubes) when measur-ing the solution volume as this determines theamount of CsCl to be added. If the amount ofadded CsCl is incorrect, the position of thebands will be high (if too much was added) orlow (if too little was added). Ideally the bandsshould appear slightly above the center of thetube. It is also important to allow sufficient timefor the establishment of the gradient duringcentrifugation. If the bands appear diffuse atthe end of the run, resolution is not adequateand centrifugation should be continued. In agradient that has achieved equilibrium, bandsare well defined.

Occasionally an ultracentrifuge tube breaksand its contents leak into the rotor compart-ment. This can occur if the tube is defective orif it is not filled or sealed properly. If most ofthe solution remains in the rotor compartmentit can be pipetted into a clean tube, topped upwith CsCl/TE solution, and centrifuged again.The quality of the DNA should not be affected,


1.7.13


although the yield will be lower due to loss ofmaterial. The rotor should be rinsed with warmwater if it comes in contact with CsCl solution.Cesium chloride is very corrosive and can causepitting and weakening of the rotor. The centri-fuge chamber should be inspected after everyrun, and cleaned with warm water and driedthoroughly if red stains are evident.

If the CsCl precipitates, as evidenced by alarge, white, crystalline pellet observed whenprecipitating plasmid DNA in the final steps,warm the solution to room temperature andcentrifuge it at room temperature. If this prob-lem persists, be certain the solution is diluted3-fold with TE buffer before adding ethanol.

Plasmid DNA purification by PEG precipi-tation. The purity of plasmid DNA obtained bypolyethylene glycol (PEG) precipitation ofcrude bacterial lysate depends on the amountof chromosomal DNA remaining in the solu-tion when the PEG solution is added. Steps 1to 5 of Alternate Protocol 3 remove any remain-ing traces of chromosomal DNA. These stepsare not necessary for PEG precipitation to suc-ceed. For size fractionation by PEG to be effec-tive, the concentration of DNA in the crudelysate must be >10 µg/ml. This is not a concernwhen applied to plasmid DNA purification,where the concentrations should be orders ofmagnitude greater than that figure.

Plasmid DNA purification by anion-ex-change or size-exclusion chromatography.The columns available in kit form from a num-ber of manufacturers are quite reliable. Almostall necessary reagents are provided, includingcommon buffers such as TE buffer. Because thecomposition of the matrix is undisclosed, it isimpossible to evaluate the procedure carefullyand attempt to optimize it. Therefore, users ofkits are strongly encouraged to follow themanufacturer’s recommended procedures. Ad-ditional discussion of one type of anion-ex-change procedure (Qiagen) can be found in theCommentary of UNIT 2.1B.

A major drawback to some prepared col-umns is the limited capacity of the matrix. A500-ml culture of plasmid-containing bacteriacan often yield 2 to 8 mg of plasmid DNA inthe crude lysate. Some columns routinely yieldonly 500 to 1000 µg purified plasmid DNA,which is adequate for most purposes. Recover-ing the column flowthrough and rechroma-tographing it is the most practical method ofincreasing recovery of plasmid DNA. Not allcolumns can be reused, however, and recoverymay require use of additional columns. Alter-

natively, smaller culture volumes can be usedas suggested. If optimal recovery of DNA isdesired, CsCl/ethidium bromide centrifugationor PEG precipitation should be used.

DNA obtained from chromatographic puri-fication of plasmid DNA is comparable in qual-ity to that prepared by the other methods. It isof sufficient purity for virtually any procedurefor which it can be used. For example, plasmidDNA prepared using Qiagen-tips is at leastequivalent in purity to that obtained followingtwo rounds of CsCl-gradient centrifugation. Itis suitable for all applications from cloning totransfection, radioactive and fluorescent se-quencing, and gene therapy research. Qiagenalso offers an endotoxin-free plasmid DNA kitwhich yields plasmid DNA containing lowerlevels of endotoxins than DNA purified by tworounds of CsCl centrifugation, which is essen-tial in gene therapy studies.

The two most frequent contaminants arechromosomal DNA and high-molecular-weight RNA. These contaminants may be de-tected by the presence of large, diffuse ethidiumbromide–binding material in agarose gel elec-trophoresis of purified plasmid DNA. To pre-vent such contamination, follow the manufac-turer’s suggestions for the use of RNase.

Only common laboratory equipment is re-quired for chromatographic purification ofplasmid DNA. Another consideration withcommercial kits is the large amount of packag-ing material and waste. In addition to the plasticcolumns and excess packaging, kits containstandard reagents supplied in plastic bottles.These reagents are solutions of buffers, salts,ethanol, and detergent—all of which can be,and usually are, prepared in the lab. Most sup-pliers will provide the column without the re-agents. However, the major advantage of usingprepared anion-exchange column kits includesthe absence of organic extractions and exposureto toxic chemicals such as phenol, chloroform,ethidium bromide, and CsCl.

The toxicity of DNA prepared by severalmethods has been assessed by performing abiological assay. Crude lysate from a 1-literculture of plasmid-containing bacteria was pre-pared by the alkaline lysis procedure and di-vided into four equal aliquots. The aliquotswere then subjected to purification by CsCl/ethidium bromide centrifugation, PEG precipi-tation, or chromatography on Qiagen andpZ523 columns. Purified plasmid DNA wasinjected into Drosophila embryos, and the fre-quency of germline transformation and killing


1.7.14


of injected embryos was determined. No sig-nificant differences attributable to the methodof purification were observed.

Anticipated ResultsMost plasmids currently used are derivatives

of the pUC series (Fig. 1.5.2). These plasmidscontain an origin of replication significantlymore efficient than that of the previous genera-tion of pBR322-derived plasmids. This allowsrecovery of 1 to 5 mg of plasmid DNA (free ofcontaminating bacterial products) from a 500-ml culture following any of the crude lysatepreparation methods or PEG precipitation. Pu-rification by CsCl/ethidium bromide densitygradient centrifugation yields 75% to 90% ofthe amount of plasmid DNA obtained usingPEG precipitation. Yields obtained from col-umn chromatography are limited by the capac-ity of the column and are generally <1 mg.

Time ConsiderationsCell growth and concentration require one

overnight growth period to collect the starting5-ml culture and most of the next day for itsoutgrowth and concentration. Crude lysatepreparation can be completed in 90 min usingalkaline lysis, ∼35 min using the boilingmethod, and 2.5 to 3 hr using Triton lysis.Plasmid DNA purification by CsCl/ethidiumbromide density gradient centrifugation takes4 hr to 3 days depending upon the quality ofplasmid DNA desired and the type of centrifugeand rotor used. PEG precipitation of the crudelysate can yield pure plasmid DNA in as littleas 2 hr; however, complete recovery may re-quire 13 to 16 hr. Column chromatographytakes 30 to 90 minutes. High-quality plasmidDNA recovery from an E. coli strain containingthe desired plasmid using any combination ofthese procedures takes 1 day to 1 week.

In addition to the total time necessary toobtain pure plasmid DNA, it is important toconsider the hands-on time required for indi-vidual steps of the different procedures. PEGprecipitation can yield pure plasmid DNA 4 or5 hr after harvesting cells but requires directattention and manipulations every 10 to 30 min.Purification using CsCl/ethidium bromide cen-trifugation takes 6 to 18 hr after harvestingcells, but the final 4-5 hr are taken up by acentrifugation step that requires no direct atten-tion. Column chromatography can yield puri-fied DNA within 3 hr of harvesting cells. WithQiagen protocols, plasmid purification timescan also be reduced by using a Qiafilter to clearthe bacterial lysate. In addition, most of these

columns operate by gravity flow for maximumhandling convenience; the columns never rundry. Therefore, if pure plasmid DNA is neededthe same day the large culture is harvested, it isperhaps best to prepare crude lysates by alka-line lysis and purify plasmid DNA by PEGprecipitation, collecting the PEG precipitateafter ∼1 hr at 0°C, or by chromatography. If ithas been a long day and the priority is to gohome, crude lysate can be prepared by theboiling method and plasmid DNA purified bythe CsCl/ethidium bromide centrifugation. Thevariety of methods and the opportunity to in-terrupt them at different steps (such as at theprecipitation steps) facilitate selection of meth-ods most convenient for specific work situ-ations.

Literature CitedClewell, D.B. and Helinski, D.R. 1970. Properties

of a deoxyribonucleic acid-protein relaxationcomplex and strand specificity of the relaxationevent. Biochemistry 9:4428-4440.

Clewell, D.B. and Helinski, D.R. 1972. Nature ofColE1 plasmid replication in Escherichia coli inthe presence of chloramphenicol. J. Bacteriol.110:667-676.

Tartoff, K.D. and Hobbs, C.A. 1987. Improved me-dia for growing plasmid and cosmid clones. BRLFOCUS 9:2-12.

Key ReferencesBirnboim, H.C. 1983. A rapid alkaline extraction

method for the isolation of plasmid DNA. Meth-ods Enzymol. 100:243-255.

Birnboim, H.C. 1988. Citation classic. Current Con-tents (Life Sciences) 31(45):12.

Birnboim, H.C. and Doly, J. 1979. A rapid alkalineextraction method for screening recombinantplasmid DNA. Nucl. Acids Res. 7:1513-1523.

These three references describe preparation ofcrude lysate by alkaline lysis.

Holmes, D.S. and Quigley, M. 1981. A rapid boilingmethod for the preparation of bacterial plasmids.Anal. Biochem. 114:193-197.

Describes preparation of crude lysate by boiling.

Clewell and Helinski, 1970, 1972. See above.

These two references describe preparation of crudelysate by Triton X-100 lysis.

Radloff, R., Bauer, W., and Vinograd, J. 1967. Adye-buoyant-density method for the detectionand isolation of closed circular duplex DNA: Theclosed circular DNA in HeLa cells. Proc. Natl.Acad. Sci. U.S.A. 57:1514-1521.

Describes plasmid DNA purification by CsCl/ethid-ium bromide centrifugation.


1.7.15


Lis, J.T. 1980. Fractionation of DNA fragments bypolyethylene glycol induced precipitation.Methods Enzymol. 65:347-353.

Lis, J.T. and Schleif, R. 1975. Size fractionation ofdouble-stranded DNA by precipitation withpolyethylene glycol. Nucl. Acids Res. 2:383-389.(Correction: the photograph of Fig. 1 should beinterchanged with the photograph of Fig. 2. Lisand Schleif, 1975, Nucl. Acids Res. 2:757.)

These two references describe plasmid DNA purifi-cation by PEG precipitation.

Contributed by J.S. HeiligUniversity of ColoradoBoulder, Colorado

Karen L. ElbingClark and Elbing LLPBoston, Massachusetts

Roger BrentMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts


1.7.16


UNIT 1.8Introduction of Plasmid DNA into Cells

Transformation of E. coli can be achieved using any of the four protocols in this unit. Thefirst method (see Basic Protocol 1) using calcium chloride gives good transformationefficiencies, is simple to complete, requires no special equipment, and allows storage ofcompetent cells. The one-step method (see Alternate Protocol 1) is considerably fasterand also gives good transformation efficiencies (although they are somewhat lower).However, because it was developed relatively recently, its reproducibility and reliabilityare not as well established.

If considerably higher transformation efficiencies are needed, the third method (see BasicProtocol 2) using electroporation should be followed. Although this procedure is simple,fast, and reliable, it requires an electroporation apparatus. As in the calcium chlorideprotocol, prepared cells can be stored. The final method described (see Alternate Proto-col 2) describes an adaptation based on electroporation that allows direct transfer of vectorDNA from yeast into E. coli.

BASICPROTOCOL 1

TRANSFORMATION USING CALCIUM CHLORIDE

Escherichia coli cells are grown to log phase. Cells are concentrated by centrifugationand resuspended in a solution containing calcium chloride. Exposure to calcium ionsrenders the cells able to take up DNA, or competent. Plasmid DNA is mixed with the cellsand presumably adheres to them. The mixture of DNA and cells is then heat shocked,which allows the DNA to efficiently enter the cells. The cells are grown in nonselectivemedium to allow synthesis of plasmid-encoded antibiotic resistance proteins, then platedon antibiotic-containing medium to allow identification of plasmid-containing colonies.

Materials

Single colony of E. coli cellsLB medium (UNIT 1.1)CaCl2 solution (see recipe), ice coldLB plates (UNIT 1.1) containing ampicillin (Table 1.4.1)Plasmid DNA (UNITS 1.6 & 1.7)

Chilled 50-ml polypropylene tubesBeckman JS-5.2 rotor or equivalent42°C water bath

Additional reagents and equipment for growth of bacteria in liquid media (UNIT 1.2)

NOTE: All materials and reagents coming into contact with bacteria must be sterile.

Prepare competent cells1. Inoculate a single colony of E. coli cells into 50 ml LB medium. Grow overnight at

37°C with moderate shaking (250 rpm; see UNIT 1.2).

Alternatively, grow a 5-ml culture overnight in a test tube on a roller drum.

2. Inoculate 4 ml of the culture into 400 ml LB medium in a sterile 2-liter flask. Growat 37°C, shaking at 250 rpm, to an OD590 of 0.375.

This procedure requires that cells be growing rapidly (early- or mid-log phase). Accord-ingly, it is very important that the growing cells have sufficient air. A 1-liter baffle flask canbe used instead of the 2-liter flask. Overgrowth of culture (beyond OD590 of 0.4) decreasesthe efficiency of transformation.

Supplement 37

Contributed by Christine E. Seidman, Kevin Struhl, Jen Sheen, and Timm JessenCurrent Protocols in Molecular Biology (1997) 1.8.1-1.8.10Copyright © 1997 by John Wiley & Sons, Inc.

1.8.1


3. Aliquot culture into eight 50-ml prechilled, sterile polypropylene tubes and leave thetubes on ice 5 to 10 min.

Cells should be kept cold for all subsequent steps.

Larger tubes or bottles can be used to centrifuge cells if volumes of all subsequent solutionsare increased in direct proportion.

4. Centrifuge cells 7 min at 1600 × g (3000 rpm in JS-5.2), 4°C. Allow centrifuge todecelerate without brake.

We have not attempted to determine whether deceleration without braking is critical to thisprocedure. However, we do not use the brake for this step or for the subsequent centrifu-gation steps.

5. Pour off supernatant and resuspend each pellet in 10 ml ice-cold CaCl2 solution.

Resuspension should be performed very gently and all cells kept on ice.

6. Centrifuge cells 5 min at 1100 × g (2500 rpm), 4°C. Discard supernatant andresuspend each pellet in 10 ml ice-cold CaCl2 solution. Keep resuspended cells onice for 30 min.

7. Centrifuge cells 5 min at 1100 × g, 4°C. Discard supernatant and resuspend eachpellet in 2 ml ice-cold CaCl2 solution.

It is important to resuspend this final pellet well. The suspension may be left on ice forseveral days. For many strains (e.g., DH1) competency increases with increasing time onice, and reaches a maximum at 12 to 24 hr. This is not true for MC1061 cells, which shouldbe frozen immediately.

8. Dispense cells into prechilled, sterile polypropylene tubes (250-µl aliquots areconvenient). Freeze immediately at −70°C.

Assess competency of cells9. Use 10 ng of pBR322 to transform 100 µl of competent cells according to the steps

provided below. Plate appropriate aliquots (1, 10, and 25 µl) of the transformationculture on LB/ampicillin plates and incubate at 37°C overnight.

10. Calculate the number of transformant colonies per aliquot volume (µl) × 105: this isequal to the number of transformants per microgram of DNA.

Transformation efficiencies of 107 to 108 and 106 to 107 are obtained for E. coli MC1061and DH1, respectively. Competency of strains decreases very slowly over months of storagetime.

Transform competent cells11. Aliquot 10 ng of DNA in a volume of 10 to 25 µl into a sterile 15-ml round-bottom

test tube and place on ice.

Plasmid DNA can be used directly from ligation reactions. When this is done, more DNAis usually used. However, if there is >1 �g of DNA in the ligation reaction, or if the ligationreaction is from low gelling/melting temperature agarose, it is wise to dilute the ligationmix (see UNIT 3.16).

12. Rapidly thaw competent cells by warming between hands and dispense 100 µlimmediately into test tubes containing DNA. Gently swirl tubes to mix, then placeon ice for 10 min.

Competent cells should be used immediately after thawing. Remaining cells should bediscarded rather than refrozen.


1.8.2

Introduction ofPlasmid DNA into

Cells

13. Heat shock cells by placing tubes into a 42°C water bath for 2 min.

Alternatively, incubate at 37°C for 5 min.

14. Add 1 ml LB medium to each tube. Place each tube on a roller drum at 250 rpm for1 hr at 37°C.

15. Plate aliquots of transformation culture on LB/ampicillin or other appropriateantibiotic-containing plates.

It is convenient to plate several different dilutions of each transformation culture. Theremainder of the mixture can be stored at 4°C for subsequent platings.

16. When plates are dry, incubate 12 to 16 hr at 37°C.

ALTERNATEPROTOCOL 1

ONE-STEP PREPARATION AND TRANSFORMATIONOF COMPETENT CELLS

This procedure is considerably easier than Basic Protocol 1 because it eliminates the needfor centrifugation, washing, heat shock, and long incubation periods (Chung et al., 1989).Moreover, competent cells made by this simple procedure can be directly frozen at −70°Cfor long-term storage. A variety of strains can be made competent by this procedure, andthe transformation frequency can be as high as that achieved by Basic Protocol 1.However, frequency is considerably lower than can be obtained by electroporation.


2× transformation and storage solution (TSS; see recipe), ice coldLB medium (UNIT 1.1) containing 20 mM glucose

1. Dilute a fresh overnight culture of bacteria 1:100 into LB medium and incubate at37°C until the cells reach an OD600 of 0.3 to 0.4.

The procedure will work if cells are harvested at other stages of the growth cycle (includingstationary phase), but with reduced efficiency.

2. Add a volume of ice-cold 2× TSS equal to that of the cell suspension, and gently mixon ice.

For long-term storage, freeze small aliquots of the suspension in a dry ice/ethanol bath andstore at −70°C. To use frozen cells for transformation, thaw slowly and then use immedi-ately.

Cells can also be used if pelleted by centrifugation 10 min at 1000 × g, 4°C, and this mayincrease the frequency of transformation (according to Chung et al., 1989). Discardsupernatant and resuspend cell pellet at one-tenth of original volume in 1× TSS (preparedby diluting 2× TSS). Proceed with transformation as in step 3.

3. Add 100 µl competent cells and 1 to 5 µl DNA (0.1 to 100 ng) to an ice-coldpolypropylene or glass tube. Incubate 5 to 60 min at 4°C.

As is the case for related procedures, the transformation frequency as measured bytransformants/�g DNA is relatively constant at amounts of DNA <10 ng. However, thefrequency decreases at higher concentrations. The time of incubation at 4°C is relativelyunimportant.

4. Add 0.9 ml LB medium containing 20 mM glucose and incubate 30 to 60 min at 37°Cwith mild shaking to allow expression of the antibiotic resistance gene. Selecttransformants on appropriate plates.

It is unnecessary to heat shock the transformation mixture. The expected transformationfrequency should range between 106 and 107 colonies/�g DNA.


1.8.3


BASICPROTOCOL 2

HIGH-EFFICIENCY TRANSFORMATION BY ELECTROPORATION

Electroporation with high voltage is currently the most efficient method for transformingE. coli with plasmid DNA. The procedure described may be used to transform freshlyprepared cells or to transform cells that have been previously grown and frozen. Withfreshly grown cells, it routinely gives more than 109 bacterial transformants per micro-gram of input plasmid DNA.

Materials

Single colony of E. coli cellsLB medium (UNIT 1.1)H2O, ice cold10% glycerol, ice coldSOC medium (see recipe)LB plates (UNIT 1.1) containing antibiotics (Table 1.4.1)

1-liter centrifuge bottle, 50-ml narrow-bottom polypropylene tube, andmicrocentrifuge tubes, chilled ice cold

Beckman J-6M centrifuge (or equivalent)Beckman JS-4.2 rotor (or equivalent) and adaptors for 50-ml narrow-bottom tubesElectroporation apparatus with a pulse controller or 200- or 400-ohm resistorChilled electroporation cuvettes, 0.2-cm electrode gap

Additional reagents and equipment for growth of bacteria in liquid media (UNIT 1.2)

NOTE: All materials and reagents coming into contact with bacteria must be sterile.

Prepare the cells1. Inoculate a single colony of E. coli cells into 5 ml LB medium. Grow 5 hr to overnight

at 37°C with moderate shaking (see UNIT 1.2).

2. Inoculate 2.5 ml of the culture into 500 ml LB medium in a sterile 2-liter flask. Growat 37°C, shaking at 300 rpm, to an OD600 of ∼0.5 to 0.7.

Best results are obtained by harvesting cells at an OD600 of ∼0.5 to 0.6.

3. Chill cells in an ice-water bath 10 to 15 min and transfer to a prechilled 1-litercentrifuge bottle.

Cells should be kept at 2°C for all subsequent steps.

4. Centrifuge cells 20 min at 4200 rpm in Beckman J-6M, 2°C.

5. Pour off supernatant and resuspend the pellet in 5 ml ice-cold water. Add 500 mlice-cold water and mix well. Centrifuge cells as in step 4.

6. Pour off supernatant immediately and resuspend the pellet by swirling in remainingliquid.

Because the pellet is very loose, the supernatant must be poured off immediately. The pelletcan be made tighter by substituting ice-cold sterile HEPES (1 mM, pH 7.0) for the ice-coldwater in step 5.

7. Add another 500 ml ice-cold water, mix well, and centrifuge again as in step 4.

8. Pour off supernatant immediately and resuspend the pellet by swirling in remainingliquid.


1.8.4


Cells

9a. If fresh cells are to be used for electroporation, place suspension in a prechilled,narrow-bottom, 50-ml polypropylene tube, and centrifuge 10 min at 4200 rpm inBeckman J-6M centrifuge with JS-4.2 rotor and adaptors, 2°C.

Fresh cells work better than frozen cells.

Estimate the pellet volume (usually ∼500 �l from a 500-ml culture) and add an equalvolume of ice-cold water to resuspend cells (on ice). Aliquot 50- to 300-�l cells intoprechilled microcentrifuge tubes. The cell density is ∼2 × 1011/ml.

9b. If frozen cells are to be used for electroporation, add 40 ml ice-cold 10% glycerol tothe cells and mix well. Centrifuge cells as described in step 9a.

Estimate the pellet volume and add an equal volume of ice-cold 10% glycerol to resuspendcells (on ice). Place 50- to 300-�l aliquots of cells into prechilled microcentrifuge tubesand freeze on dry ice (not in liquid nitrogen). Store at −80°C.

Prolonged incubation of cells in ice-water at all stages can increase transformationefficiency of some strains, such as BW313/P3 and MC1061/P3, >3-fold.

Transform the cells10. Set the electroporation apparatus to 2.5 kV, 25 µF. Set the pulse controller to 200 or

400 ohms.

The pulse controller is necessary when high-voltage pulses are applied over short gaps inhigh-resistance samples (see Background Information).

11. Add 5 pg to 0.5 µg plasmid DNA in 1 µl to tubes containing fresh or thawed cells(on ice). Mix by tapping the tube or by swirling the cells with the pipettor.

12. Transfer the DNA and cells into a cuvette that has been chilled 5 min on ice, shakeslightly to settle the cells to the bottom, and wipe the ice and water from the cuvettewith a Kimwipe.

The volume of DNA added to the cells should be kept small. Adding DNA up to one-tenthof the cell volume will decrease the transformation efficiency 2- to 3-fold. Also, since theresistance of the sample should be high, make sure that addition of the DNA to the cellsdoes not increase the total salt concentration in the cuvette by >1 mM.

13. Place the cuvette into the sample chamber.

If using a homemade apparatus, connect the electrodes to the cuvette.

14. Apply the pulse by pushing the button or flipping the switch.

15. Remove the cuvette. Immediately add 1 ml SOC medium and transfer to a sterileculture tube with a Pasteur pipet. Incubate 30 to 60 min with moderate shaking at37°C.

If the actual voltage and time constant of the pulse are displayed on the electroporationapparatus, check this information. Verify that the set voltage was actually delivered, andrecord the time constant of the pulse so that you may vary it later if necessary (see CriticalParameters).

16. Plate aliquots of the transformation culture on LB plates containing antibiotics.


1.8.5


ALTERNATEPROTOCOL 2

DIRECT ELECTROPORETIC TRANSFER OF PLASMID DNA FROMYEAST INTO E. COLI

The use of “shuttle vectors”—plasmids that can be grown successfully in at least twodifferent organisms—facilitates the transfer of DNA between, for example, yeast and E.coli. In this adaptation of the electroporation protocol, plasmid DNA from a shuttle vectoris transformed directly from yeast into E. coli. Components of the interaction trap/two-hybrid system (UNIT 20.1) are used as an example in this protocol. The transfer and selectionof a “prey” plasmid from the yeast strain EGY48 into the E. coli strain KC8 is describedhere, but the approach can be adapted for use with other yeast and E. coli strains.


Single colony of E. coli KC8 cells (UNIT 20.1)Streak colony of Trp− plasmid–harboring EGY48 yeast cells on Gal/Raff/Xgal/CM

plates (UNIT 20.1), no older than 2 weeksM9 minimal medium and plates (UNIT 1.1) containing 100 µg/ml ampicillin (Table

1.4.1) and standard concentrations of leucine, histidine, and uracil

Additional reagents and equipment for growth and manipulation of yeast (UNIT 13.2)and for plasmid DNA miniprep (UNIT 1.6) or PCR (UNIT 15.1)

1. Prepare electrocompetent KC8 cells (see Basic Protocol 2, steps 1 to 9a), resuspend-ing the final cell pellet in ice-cold water to obtain an OD600 of 100.

Fresh KC8 cells work better in this electroporation method than frozen ones.

To measure OD, dilute 5 �l of the cell suspension with water to 1 ml and measure the OD600.If necessary add more water to the suspension to get an OD600 of 100.

2. Distribute 65-µl aliquots of the electrocompetent E. coli KC8 cells into ice-coldmicrocentrifuge tubes.

3. With a sterile wooden or plastic stick, scrape off ∼10 µl of yeast from a streak colonyof EGY48 harboring the respective “prey” plasmid derivative of pJG4-5 and grownon Gal/Raff/Xgal/CM plates. Resuspend the yeast cells in the KC8 suspension byswirling the stick used for scraping off the cells.

Avoid scraping off plate medium when collecting the yeast streak cells.

Keep the microcentrifuge tube on ice as much as possible. Try to get an even distributionof the two cell types but do not vortex. Yeast grown on plates other than Gal/Raff/Xgal/CMwill probably work as well; do not worry if the yeast colony used is blue.

4. Set the electroporation apparatus to 1.5 kV, 25 µF, and the pulse controller to 100ohms. Transfer the cell suspension into a 0.2-cm cuvette that has been chilled 5 minon ice, shake slightly to settle the cells to the bottom, and wipe the ice and water fromthe cuvette with a Kimwipe.

The use of Pasteur pipettes will facilitate placing the cell suspension at the bottom of thecuvette. Avoid any air bubbles.

5. Place the cuvette in the sample chamber of the apparatus and pulse. Take the cuvetteout and place it on ice for ≥45 sec. Meanwhile, change the settings in preparation forthe second pulse.

The expected time constant for the first pulse is 2.2 to 2.4 msec.

6. Set the electroporation apparatus to 2.5 kV, 25 µF, and the pulse controller to 200ohms. Wipe the cuvette again, place it in the sample chamber, and pulse.

The expected time constant for the second pulse is 4.2 to 4.8 msec.


1.8.6


Cells

7. Remove the cuvette, immediately add 1 ml LB medium, and transfer the suspensioninto a microcentrifuge tube. Incubate 45 min at room temperature.

Incubation of the suspension after electroporation for 1 hr at 37°C decreases the yield oftransformants, probably due to prolonged adhesion of the E. coli cells to the yeast celldebris.

8. Spread 150 µl of the suspension evenly onto M9 minimal medium plates containing100 µg/ml ampicillin and leucine, histidine, and uracil. Incubate ≥24 hr at 37°C.

A slight yeast background might appear on the plates but single E. coli colonies are easyto pick. Between 50 and 200 KC8 colonies have been obtained per plate employing 150 �lout of the 1 ml LB suspension.

9. Pick a single KC8 colony, inoculate it into 1.5 to 5 ml M9 minimal medium (Leu+,His+, Ura+, 100 µg/ml Amp) or LB (100 µg/ml Amp) and grow at 37°C. Harvest atan appropiate OD to prepare miniprep DNA (UNIT 1.6) or perform PCR analysis(UNIT 15.1).

Using M9 minimal medium to grow KC8 in liquid culture is an additional safety measurebut not absolutely necessary. It ensures isolation of the plasmid whose marker complementsthe auxotrophic defect in KC8; in addition, slightly increased plasmid copy number andimproved DNA quality have been reported.


CaCl2 solution60 mM CaCl2

15% glycerol10 mM PIPES [piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)], pH 7Filter sterilize using a disposable filter unit, or autoclaveStore at room temperature (stable for years)

SOC medium0.5% yeast extract2% tryptone10 mM NaCl2.5 mM KCl10 mM MgCl2

10 mM MgSO4

20 mM glucoseStore at room temperature (stable for years)

Transformation and storage solution (TSS), 2×Dilute sterile (autoclaved) 40% (w/v) polyethylene glycol (PEG) 3350 to 20% PEGin sterile LB medium containing 100 mM MgCl2. Add dimethyl sulfoxide (DMSO)to 10% (v/v) and adjust to pH 6.5.

COMMENTARY

Background Information

Calcium and one-step transformationTransformation of E. coli was first described

by Mandel and Higa (1970). Subsequent mod-ifications to improve transformation efficien-cies have included prolonged exposure of cellsto CaCl2 (Dagert and Ehrlich, 1974), substitu-tion of calcium with other cations such as Rb+

(Kushner, 1978), Mn2+, and K+, and additionof other compounds such as dimethyl sulfox-ide, dithiothreitol, and cobalt hexammine chlo-ride (Hanahan, 1983). Basic Protocol 1 givenhere provides good transformation efficiencies,permits long-term storage of competent cells,and is relatively uncomplicated to perform.Variations on this protocol can be obtainedfrom the references provided. Alternate Proto-


1.8.7


col 1, a one-step preparation and transforma-tion procedure, is considerably faster.

Transformation by electroporationElectroporation has become a valuable tech-

nique for transfer of nucleic acids into eu-karyotic and prokaryotic cells. The method canbe applied to many different E. coli strains andto other gram-negative and gram-positive bac-teria.

In this technique, a high-voltage electricfield is applied briefly to cells, apparently pro-ducing transient holes in the cell membranethrough which plasmid DNA enters (Shigek-awa and Dower, 1988). The field strength usedfor mammalian cell and plant protoplast elec-troporation is usually 0.5 to 1 kV/cm. The fieldstrength needed for high-efficiency transfor-mation of E. coli is much greater, usually ∼12.5kV/cm. Under these conditions up to 1010 trans-formants/µg plasmid DNA have been reported(Calvin and Hanawalt, 1988; Miller et al., 1988;Dower et al., 1988). Recently, field strength upto 8 kV/cm was also used successfully to elec-troporate both mammalian cells and plant pro-toplasts.

The capacitor discharge circuit of the elec-troporation apparatus typically generates anelectrical pulse with an exponential decaywaveform. The voltage across the electrodesrises rapidly to a peak voltage, which thendeclines over time as follows:

Vt = V0 [e−t/T]

where Vt = voltage at a given time t after thetime of V0, V0 = initial voltage, t = time (sec),T = pulse time constant = RC, R = resistance ofcircuit (ohms); and C = capacitance of circuit(Farads).

The pulse time constant is ∼5 to 10 msec forelectroporating E. coli cells and ranges from 5µsec to 50 msec for higher eukaryotic cells.

The pulse controller contains a number ofdifferent-sized resistors, any one of which isplaced in parallel with the sample, and oneresistor of fixed (20-ohm) resistance, which isplaced in series with the sample. The resistorplaced in parallel with the sample (usually 200or 400 ohms) swamps out the effect of changesin the resistance of the sample on the totalresistance of the circuit, thus determining thetotal resistance across the capacitor, and con-trolling the time constant (T) of the capacitordischarge. The 20-ohm resistor in series withthe sample protects the circuitry by limiting thecurrent should a short circuit (arc) occur andthe capacitor discharge instantly.

The pulse controller is required when high-voltage electroporation pulses are delivered tohigh-resistance samples across narrow elec-trode gaps. In this procedure, the resistance ofwashed E. coli in the 0.2-cm cuvette is ∼5000ohms. A pulse controller is not necessary whensamples of low (<1000-ohm) resistance areused, for example for electroporation of mam-malian cells suspended in PBS.

Direct transfer of plasmid DNA from yeastinto E. coli

Alternate Protocol 2 presents an applicationof electrophoretic transformation whereby a“shuttle vector” (a vector designed to be usedin at least two different organisms to facilitateinterspecies transfer of DNA) can be directlytransferred between two species. Shuttle vec-tors have become increasingly popular in recentyears, with those designed to facilitate thetransfer of plasmid DNA between yeast and E.coli proving to be particularly useful. With thewidespread use of the two-hybrid system (orinteraction trap; see UNIT 20.1), transfer of plas-mid DNA from yeast into E. coli using shuttlevectors has become a common task. As analternative to rescuing a shuttle plasmid from ayeast clone and subsequently transforming anappropriate E. coli strain, the procedure de-scribed in Alternate Protocol 2 bypasses theneed for plasmid isolation and offers a one-stepmethod to obtain the same result. The directtransfer method was first mentioned by Marciland Higgins (1992) and further modified byKaren Clemens (NIH, Bethesda, Md., pers.comm.). Alternate Protocol 2 comprises an op-timization of the procedure and an adaptationto the interaction trap; however, although out-lined for that specific case, the procedure isgenerally applicable to other yeast and E. colistrains. As presented, this protocol accompl-ishes the transfer of a “prey” plasmid from theyeast strain EGY48, designed to be used ininteractor hunts, into the E. coli strain KC8.EGY48 contains three different plasmids (bait,prey, and lacZ reporter), all of which conferampicillin resistance if transferred into E. coli.Therefore, the TRP1 selectable marker of thenew interactor (prey plasmid), which comple-ments the Trp− phenotype of EGY48 as well asKC8, is used to rescue the plasmid. During theelectroporation procedure the yeast cells aredestroyed in the first pulse and the liberatedplasmids transformed into E. coli KC8 cells inthe second pulse. Selection of the prey plasmidis achieved by choosing the correct medium onwhich to plate the transformed KC8.


1.8.8


Cells

Critical Parameters

Calcium transformationIn Basic Protocol 1, preparation of compe-

tent cells with a high transformation efficiencyis thought to depend on (1) harvesting bacterialcultures in logarithmic phase of growth, (2)keeping cells on ice throughout the procedure,and (3) prolonged CaCl2 exposure.

At least 30 min of growth in nonselectivemedium (outgrowth) after heat shock is necessaryfor plasmids containing the pBR322 tetracylineresistance promoter and gene to express enoughof the protein to allow the cells to form colonieswith an efficiency of 1 on tetracycline plates.Cells expressing the common plasmid-encodedampicillin resistance (β-lactamase) gene maynot require such prolonged outgrowth to formcolonies on ampicillin plates. When an am-picillin-resistant plasmid is used, transforma-tion mixtures should be diluted so that trans-formed colonies arise at a relatively low density(≤500 cells/plate). Otherwise, the β-lactamasespresent in the colonies may lower the ampicillinlevel in the plate near them, and permit growthof weakly ampicillin-resistant satellite colo-nies. This problem can be ameliorated if carbe-nicillin (a related antibiotic slightly less sensi-tive to destruction by the pBR322 β-lactamase)is substituted for ampicillin in the medium.Carbenicillin should be used at a concentrationof 50 to 100 µg/ml.

Usually only 3% to 10% of cells are compe-tent to incorporate plasmid DNA. Transforma-tion frequencies decrease with increasing plas-mid size (Hanahan, 1983). The number of tran-sformants obtained usually increases linearlywith increasing numbers of plasmid moleculesup to a point, reached at ∼10 ng DNA/100 µlcompetent cells in the procedure given here.After this point the number of transformantsdoes not increase linearly with increasing num-bers of plasmid DNA molecules.

Transformation by electroporationAlthough the procedure works with cells

grown to many different densities, best resultsare obtained when cells are harvested at anOD600 of 0.5 to 0.6. After the cells are centri-fuged in water, the cell pellet is very loose andthe supernatant should be poured off as quicklyas possible to prevent a big loss in yield; toachieve this, it is best to handle no more thantwo centrifuge bottles simultaneously. Fresh

cells in either water or 10% to 20% glycerolusually work better than frozen cells.

As described above, the relevant parametersfor exponential electroporation pulses are thetime constant of the exponential curve (howlong the pulse lasts) and the initial voltage orfield strength (how strong the pulse is). Gener-ally speaking, successful electroporation of E.coli requires long, strong pulses. In this pro-cedure the capacitance of the circuit is rela-tively large (25 µF), ensuring a relatively longpulse; fine tuning of the pulse time constant isachieved by varying the size of the resistorplaced in parallel with the sample.

The SOC medium must be added immedi-ately after electroporation. Do not let the elec-troporated cells sit in the cuvette.

In the procedure given here, the number oftransformants increases linearly with inputDNA over a very wide range (from 5 pg to500 ng). The amount of plasmid DNA addedcan be as little as 4 to 5 pg in 50 µl of cells, andas much as 0.5 µg in 300 µl of cells, withoutaffecting the transformation efficiency signifi-cantly. However, transformation efficiency fallsoff with <5 pg of DNA; in one experiment, thenumber of transformants obtained with 1 pg ofplasmid DNA was 30 times lower than with 5 pg.

The size of the DNA does not seem to beimportant for this procedure; plasmid DNA canbe as large as 14 kb without significant effectson transformation efficiency. Religated DNA(still in the ligation mix) can be transformedalmost as efficiently as intact supercoiled DNA.

The general recommendations given forelectroporation apply to the direct transfermethod as well. Although outlined for a specificcase, the procedure described in Alternate Pro-tocol 2 should be generally applicable to otheryeast and E. coli strains. The yeast streak colonyshould not be older than 2 weeks. Scrapingmedium off the plate when collecting the yeastcells should be avoided. Fresh KC8 cells workbetter than frozen ones. Incubation of the 1 mlLB suspension after electroporation at 37°C for1 hr decreases the yield of transformants, prob-ably due to prolonged adhesion of the E. colicells to the yeast cell debris. Using M9 minimalmedium to grow KC8 in liquid culture is anadditional safety measure, but is not absolutelynecessary. It ensures isolation of the plasmidwhose marker complements the auxotrophicdefect in KC8; in addition slightly increasedplasmid copy number and improved DNA qual-ity have been reported.


1.8.9


Anticipated ResultsCalcium and one-step transformation. In

Basic Protocol 1, transformation efficiencies of107 to 108 and 106 to 107/µg plasmid DNAshould be obtained for E. coli MC1061 andDH1, respectively. In Alternate Protocol 1,transformation frequencies should range from106 to 107 colonies/µg DNA.

Transformation by electroporation. UsingBasic Protocol 2, efficiencies of 2.5–14 × 1010

transformants/µg have been obtained with su-perpure pUC19 DNA (from BRL) and 6.2–12× 109 transformants/µg with home-madepUC18 DNA and cDNA libraries inMC1061/P3. In addition, 5 × 109 transfor-mants/µg have been obtained with the expres-sion plasmid CDM8 in MC1061/P3. Similarresults may be anticipated with MC1061 (seeTable 1.4.5) and with many other commonlyused lab strains. Using direct plasmid transferfrom yeast to E. coli (Alternate Protocol 2),between 50 and 200 KC8 colonies have beenobtained per plate when 150 µl out of the 1 mlLB suspension was employed.

Time ConsiderationsCalcium and one-step transformation.

Growth of competent cells from an aliquot ofan overnight culture to logarithmic phase re-quires ∼3 hr. In Basic Protocol 1, cells are thenexposed to calcium as long as overnight. Oncecompetent cells are available, transformationrequires ∼90 min for either strain. In AlternateProtocol 1, preparation and transformation ofcompetent cells requires 1 to 2 hr.

Transformation by electroporation. In Ba-sic Protocol 2, once the culture of bacterial cellsis ready to be harvested, the cells can be washedand concentrated within an hour. Electropora-tion takes only a few minutes, and growth andplating of the transformed cells should not takemore than 90 min. The time frame for directplasmid transfer (Alternate Protocol 2) is es-sentially the same as for the electroporationbasic protocol. It should be noted that using M9minimal medium slows down the growth of E.coli.

Literature CitedCalvin, N.M. and Hanawalt, P.C. 1988. High-effi-

ciency transformation of bacterial cells by elec-troporation. J. Bacteriol. 170:2796-2801.

Chung, C.T., Niemela, S.L., and Miller, R.H. 1989.One-step preparation of competent Escherichiacoli: Transformation and storage of bacterialcells in the same solution. Proc. Natl. Acad. Sci.U.S.A. 86:2172-2175.

Dagert, M. and Ehrlich, S.D. 1974. Prolonged incu-bation in calcium chloride improves competenceof Escherichia coli cells. Gene 6:23-28.

Dower, W.J., Miller, J.F., and Ragsdale, C.W. 1988.High efficiency transformation of E. coli by highvoltage electroporation. Nucl. Acids Res.16:6127-6145.

Hanahan, D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol.166:557-580.

Kushner, S.R. 1978. An improved method for trans-formation of Escherichia coli with ColEI derivedplasmids. In Genetic Engineering (H.W. Boyerand S. Nicosia, eds.) pp. 17-23. Elsevier/NorthHolland, Amsterdam.

Mandel, M. and Higa, A. 1970. Calcium-dependentbacteriophage DNA infection. J. Mol. Biol.53:159-162.

Marcil, R. and Higgins, D.R. 1992. Direct transferof plasmid DNA from yeast to E. coli by electro-poration. Nucl. Acids Res. 20:917.

Miller, J.F., Dower, W.J., and Tompkins, L.S. 1988.High-voltage electroporation of bacteria: Ge-netic transformation of Campylobacter jejuniwith plasmid DNA. Proc. Natl. Acad. Sci. U.S.A.85:856-860.

Shigekawa, K. and Dower, W.J. 1988. Electropora-tion of eukaryotes and prokaryotes: A generalapproach to the introduction of macromoleculesinto cells. BioTechniques 6:742-751.

Key References

Dower et al., 1988. See above.

The paper from which the second basic protocol wasderived, and the highest-efficiency E. coli transfor-mation by electroporation published to date.

Hanahan, 1983. See above.

An extremely thorough explanation of the parame-ters affecting transformation efficiency.

Contributed by Christine E. Seidman (calcium) and Kevin Struhl (one-step)Harvard Medical SchoolBoston, Massachusetts

Jen Sheen (electroporation)Massachusetts General HospitalBoston, Massachusetts

Timm Jessen (direct transfer)Hoechst AGFrankfurt am Main, Germany


1.8.10


Cells

SECTION IIIVECTORS DERIVED FROM LAMBDA ANDRELATED BACTERIOPHAGES

UNIT 1.9Introduction to Lambda Phages

The biology of lambda-derived vectors isextremely well understood. This introductionto the biology of λ and related phages is in-cluded to help readers use the λ vectors that areemployed in the manual (e.g., UNIT 1.11 andChapters 5 and 6), and to help readers under-stand new λ vectors that are being developed.

Lambda is a temperate phage, which means

that it can grow lytically or lysogenically. Whenλ infects a cell, it injects its DNA into the hostbacterium. When it grows lytically, it makesmany copies of its genome, packages them intonew phage particles, and lyses the host cell torelease the progeny phage. When λ grows lyso-genically, it inserts its DNA into the chromo-some of the host cell. The integrated phageDNA is replicated along with the host chromo-some (but sometimes, e.g., when the cell’sDNA is damaged, phage DNA is excised fromthe chromosome and the phage begins to grow

lytically). Lysogens are immune to superinfec-tion by additional λ phages carrying a homolo-gous immunity region. Plaques of temperatephages are turbid because they contain bothcells lysed by phage that have grown lyticallyand cells spared from further phage infectionbecause they have become lysogens (see sketch1.9A).

The λ genome is grouped into discreteblocks of related genes. This fact is quite con-venient to the molecular biologist, as it hasallowed construction of many phage-basedcloning vectors which have deletions in largestretches of DNA nonessential to lytic growth(see gray areas in sketch 1.9B). In addition tothese large areas, much of the immunity region,including the cII, cro, cI and rex genes, isinessential for lytic growth of otherwise wild-type phage; even the N gene is not essential iftR2 is deleted.

LYTIC GROWTH

Early Gene ExpressionLytic growth results in the production of

progeny phage and the eventual lysis of the hostcell. Lytic growth begins either on infection, ifa phage does not grow lysogenically, or afterthe inactivation of existing cI repressor in alysogen (see box E).

Transcription initiates from the early pro-moters, PR and PL. The PR transcript terminatesat tR1 and encodes the cro gene. The PL tran-

turbid and clear plaques

Sketch 1.9A

Nu

1

Iom

29

0

19

4

PEa

47

Ea

31

int

xis

Ea

22

be

t

cl40

1

Nu

3

A

FI

W

FII

B

Z

C

31

4

DE

Ea

8.5

U

exo

G

ga

m

T

clll

H

ssb

M

ral

L

git

K

rex

B

I

rex

A

J cro

14

6

57

60

Ea

59 6

52

04

68

22

1

cll

KV N O QSRRz

att ren

pR

pL

pRM

pRE

pI

λ+

dispensable regions of the λ genome

p′R

(continued) p′R

Sketch 1.9B

Supplement 13

Contributed by Karen Lech and Roger BrentCurrent Protocols in Molecular Biology (1987) 1.9.1-1.9.4Copyright © 2000 by John Wiley & Sons, Inc.

1.9.1


script continues to tL1 and encodes the N gene.The product of the N gene is an antiterminationfactor; that is, it allows the transcripts whichinitiate at PL or PR to proceed through tL1 andtR1 respectively. The N-antiterminated PR tran-script, which terminates at tR2, encodes the O,P, and Q genes. The O and P proteins arerequired for phage DNA replication; Q proteinis another antitermination protein, in this case,specific for transcripts initiating at a promoterlocated to the right of Q called PR′. Q-antiter-minated transcription from PR′ proceedsthrough another terminator called tR65 and thelate region, then through the head and tail geneswhich have been joined to the same transcrip-tion unit when the incoming phage DNA circu-larized, and finally terminates in b. The PR′transcript encodes proteins necessary for headand tail assembly, DNA packaging, and hostcell lysis.

This sequential expression of phage func-tions allows for the replication of the λ genome,its subsequent packaging into phage heads, andlysis of the host in the correct temporal order.

DNA ReplicationReplication during lytic growth requires

both host proteins and phage-encoded proteins.Lytic DNA replication can be divided into anearly and late phase. The early phase beginswith the injection of linear phage DNA into thebacterial cell. This DNA has, at either end,complementary, single-stranded cohesive endsgenerated by cleavage of the cos site. Thesesticky ends base pair, and are ligated by hostenzymes to yield a covalently closed circularmolecule. After supercoiling by host topoisom-

erases, this molecule is able to initiate DNAreplication. Replication begins at a unique site(called ori), and proceeds bidirectionally. Thistype of replication results first in the formationof a theta-shaped replication intermediate andlater in the production of two daughter circles.

Approximately 15 min after phage infec-tion, replication switches to the late phase. Thisis characterized by rolling-circle replication,which produces long polymers (called concate-mers) of repeated, full-length phage genomes.The gam gene product protects the concatemersfrom degradation by an exonuclease encodedby the host recB and recC genes. Concatemersare substrates for packaging (see below andUNITS 5.8 & 5.9).

Packaging and LysisLate in infection, DNA replication and pack-

aging of λ occur concurrently. Once concate-meric DNA structures are formed, they arecondensed into λ proheads (incomplete headparticles). Cos sites are recognized by λ pro-teins, and the DNA between two cos sites iscleaved from the concatemer coincident withits packaging into a single prohead (linearizingthe DNA and regenerating the cohesive ends).The remaining head proteins then assemble,and the tail, which has assembled inde-pendently, attaches to the head to form the intactphage. DNA located between two cos sites willonly be packaged in a form that can be injectedif it is between 38 and 53 kb long (a factimportant in choosing the proper vector to con-struct a library; see UNITS 5.1 and 5.2). The lastevent during lytic growth is the lysis of the host.Phage-encoded proteins disrupt the bacterialinner membrane and degrade the cell wall.

A. REQUIREMENTS FORLYTIC GROWTHN antitermination factor. Causes RNApolymerase to read through early termi-nators and expresses Q protein as well asthe replication proteins.Q antitermination factor. Causes RNApolymerase to read through tR65 and ex-presses the proteins required for head andtail assembly and host cell lysis.Replication proteins. Phage O and Pproteins work together with host proteinsto replicate DNA.Packaging and lysis proteins. Otherproteins essential for phage growth.

B. REQUIREMENTS FORDNA REPLICATIONcos ends. 12-bp cohesive overhangs thatpair and cause circularization in vivo.These are generated by cutting at cossites, which occurs during packaging.ori. Site at which DNA replication starts.O and P proteins. O protein binds DNA;P protein interacts with host-encodeddnaB protein.gam protein. Inhibits E. coli exonu-clease V (recBC nuclease) and thus pro-tects the end of the rolling circle concate-mer from degradation by this enzyme.


1.9.2

Introduction toLambda Phages

LYSOGENIC GROWTH

Gene ExpressionFollowing infection, wild-type λ phage

sometimes shuts down the majority of itsgenome and integrates into the bacterial chro-mosome; such a phage is called a prophage andthe cell that contains it is called a lysogen. Theproduct of the cI gene, λ repressor, is essentialfor lysogenic growth. This protein binds to theoperators, OL and OR, blocking transcription ofthe early genes (from PL and PR) and preventinglytic growth. Binding to OR also stimulatestranscription from PRM, a promoter that tran-scribes the cI gene. Lambda repressor thusmaintains the lysogenic state by repressing thetranscription of genes necessary for lyticgrowth as well as stimulating transcription ofitself. There are mutant strains of E. coli calledhfl−, used for the construction of λgt10 libraries(UNIT 1.11 and Chapter 5), in which a wild-typephage almost always becomes a lysogen.

The actual sequence of events leading tointegration begins (as it does during lytic

growth) with the joining of the cohesive endsand circularization of the genome. cII proteinthen binds to the Pint promoter and activates itstranscription. Int protein is made from thistranscript. Int protein catalyses a recombinationevent between the phage sequence, attP, and asite on the bacterial chromosome, attB, result-ing in integration of the phage.

Immunity Regions

A number of bacteriophages—including434, 21, 82, and 80—are related to phage λ, asevidenced by significant stretches of DNA ho-mology. Unique to each phage, however, isits immunity (imm) region (see sketch 1.9C).This region includes a number of importantregulatory sites and genes such as PL and PR,cI and cro, as well as OR and OL, and PRM. Ahost cell stably lysogenized with a particularlambdoid phage is immune to infection by asecond phage carrying the same imm region asthe lysogen, because transcription from PL andPR of the incoming phage is repressed. A lyso-

Sketch 1.9C

C. REQUIREMENTS FORPACKAGING AND LYSIScos sites. DNA is cleaved here, and DNAbetween the sites is packaged into phageheads.Nu1 and A proteins. Components of the“terminase” that cleaves at cos sites.B, C, D, E, and Nu3 proteins. Involvedin the structure or assembly of the phagehead.G, H, I, J, K, L, M, T, U, V, and Zproteins. Involved in the structure orassembly of the tail and tail fibers.R, S, and Rz proteins. S disrupts theinner membrane and the R and Rz pro-teins conspire to degrade the cell wall toachieve cellular lysis.

p RE

p RMp L

att

int xis bet N rex cro O P Qll

pRM p Rp L

λ immunityregion

OL1 OL2 OL3 OR3 OR2 OR1

OL OR c lc c lII

D. REQUIREMENTS FORLYSOGENIZATIONimm region. Contains the promoters andgenes (such as cI) that are essential forestablishment and maintenance of lyso-geny (see also section on immunity re-gion).cII and cIII proteins. cII is required forint synthesis; cIII protects cII from deg-radation by host proteases.int protein. Along with host proteins,catalyzes the integration of the phage intothe chromosome.attP site. Required for integration of thephage into the host chromosome.cos ends. Necessary for circularizationof the molecule upon infection.


1.9.3


gen can, however, be infected by a phage car-rying a different imm region. This phenomenonoccurs because the repressor encoded by a par-ticular immunity region specifically recognizesand represses only its own promoters.

InductionLambda lysogens are induced when they

excise from the host chromosome and undergolytic growth. Excision requires the phage intand xis (excise) gene products. These are syn-thesized from the N-antiterminated PL tran-script. Initiation of PL transcription occurswhen the cI product (λ repressor) is inactivated.Induction occurs spontaneously at a low fre-quency in λ lysogens. However, it is more oftendue to (1) cleavage of λ repressor during theSOS response (see below), or (2) destabiliza-tion of a thermosensitive mutant repressor atnonpermissive temperatures.

In the laboratory, the bacterial SOS responseis often induced by DNA damage caused byexposure of the cells to agents such as UV lightor mitomycin C. During this response, LexA,a bacterial repressor protein, is inactivated andvarious bacterial genes are induced. Repressorproteins of λ and related bacteriophages resem-ble LexA and are also inactivated. Some clon-

ing vectors contain a temperature-sensitive mu-tation in the cI gene (cIts). As a result, therepressor is stable and behaves like the wild-type repressor at 30°C, but is unstable at 42°C.cIts mutations allow lytic growth to be inducedsimply by increasing the temperature at whichthe lysogen is grown.

KEY REFERENCEHendrix, R., Roberts, J., Stahl, F., and Weisberg, R.

1983. Lambda II. Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY.

Contributed by Karen Lech and Roger BrentMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts

E. REQUIREMENTS FORINDUCTION OF A LYSOGENint and xis proteins. Necessary for theexcision of the phage DNA from the hostchromosome.Lytic growth requirements. Necessaryfor a productive burst of phage (see boxA).


1.9.4

Introduction toLambda Phages

UNIT 1.10Lambda as a Cloning VectorADVANTAGES OF USING LAMBDA

About the middle third of the λ genome isdispensable for lytic growth. Derivatives of phageλ that are used as cloning vectors typically containrestriction sites that flank some or all of thesedispensable genes. The major advantage to usinglambda-derived cloning vectors is that DNA canbe inserted and packaged into phages in vitro.Although the efficiency of packaging only ap-proaches 10%, phages, once packaged, formplaques on E. coli with an efficiency of 1. Whiletechniques for transformation of bacterial plas-mids have improved greatly in recent years (seeUNIT 1.8), the best routinely attainable frequenciesare about 108 transformants/µg pBR322, whichmeans that less than 1 in 1000 plasmids becometransformed into cells.

SELECTIONS FOR INSERTED DNASome phage vectors exploit the fact that λ has

a minimum genome size requirement for pack-aging, so that vector phages that do not containinserts above a minimum size are never detected.Other phage vectors utilize some genetic meansto distinguish between phages that still retain theblock of nonessential genes and recombinantphages in which the block of genes has beenreplaced with foreign DNA. Since the selectionfor the insert does not depend on the size of theinserted fragment, many of these vectors allowsmall fragments to be cloned.

Size selection. Lambda cannot be packagedinto phage heads if its genome is less than 78%or more than 105% of the length of wild-type λDNA. When a vector is chosen on the basis ofsize selection, the region dispensable for growthis cut out; the left and right arms of the phage arethen purified and ligated to cut foreign DNAunder conditions that favor concatemer forma-tion. Those phages whose left and right arms havenot been joined to an insert will have cos sites tooclose together to be packaged into viable phage.The existence of the size requirement for efficientphage packaging makes it impossible to use asingle phage vector to clone fragments of all sizes:phages that can accommodate large inserts cannotbe packaged if they contain small inserts, and viceversa. There are two special size selection tricksthat have sometimes found use in phage cloning.In the first, packaged phage are treated withEDTA or other chelating agents. The populationof surviving phage is enriched for phages withshorter than wild-type genomes. In the second,phage are plated on a mutant strain of E. coli calledpel−, which allows plaque formation by phages of

up to 110% of wild-type length and which se-verely inhibits plaque formation by phages of lessthan wild-type length.

spi− selection. red+ gam+ phages do not formplaques on a host lysogenic for the unrelated bac-teriophage P2. These phages are said to be spi+

(sensitive to P2 interference). red− gam− phages doform plaques on P2 lysogens, and so are said to bespi− (Zissler et al., 1971). P2 inhibition of red+ gam+

phage growth only occurs if the P2 is wild-type fora gene called old. Commonly used vectors likeλ2001 (Karn et al., 1984) and λEMBL3 (Frischaufet al., 1983) contain a fragment with the red+ andgam+ genes. These vectors will not form plaqueson a P2 lysogen unless the red+ gam+ fragment isdeleted and replaced with a piece of foreign DNA.

hfl− selection. The product of the λ cII geneis necessary for infecting phages to synthesizerepressor efficiently. The E. coli hflA and hflBgenes encode products whose effect is to de-crease the stability of the cII gene product(Banuett et al., 1986; Hoyt et al., 1982). Whenwild-type λ infects an hfl− strain, so muchrepressor is made that the phage almost alwayslysogenizes the infected cell, causing it to formeither no plaque or an extremely turbid plaque.Vectors like λgt10 contain a restriction site inthe cI gene. Insertion of foreign DNA into thissite inactivates the cI gene. The vector phagedoes not form plaques on the hfl− strain, butphages containing inserts instead of the cI geneform clear, normal-sized plaques.

MAPS OF LAMBDA-DERIVEDCLONING VECTORS

Maps are presented for phage cloning vec-tors that are currently used to construct librar-ies, as well as maps for not-so-modern vectorswhich were employed to construct libraries thatare still important.

The top of each page shows a simplifiedversion of the map of wild-type lambda. Thenext lines show the changes that were made inthe wild-type λ genome to generate that deriva-tive. Deletions of lambda DNA are indicatedby parentheses, and insertions of new DNA areindicated by bars. The bottom lines show tran-scripts, a genetic map, and a physical map ofthe resulting λ derivatives. The genetic nomen-clature used, especially for deletions andchanges in restriction sites, is extremely com-plicated. More explanation can be found in UNIT

1.9, in Lambda II by Hendrix et al. (1983), andin the articles cited in the figure legends.

Contributed by Karen Lech, Roger Brent, and Nina IrwinCurrent Protocols in Molecular Biology (1987) 1.10.1-1.10.11Copyright © 2000 by John Wiley & Sons, Inc.

1.10.1


Fig

ure

1.1

0.1

Wild

-typ

e la

mb

da.

R

ole

of t

he m

ajor

pha

ge t

ran-

scrip

ts (

arro

ws)

and

pha

ge g

enes

(bo

xes)

in t

he li

fecy

cle

of b

acte

-rio

phag

e λ

is d

escr

ibed

in U

NIT

S 1

.9 &

1.1

0.

Fig

ure

1.1

0.2

λEM

BL

3.

Thi

s ph

age

vect

or is

use

d fo

r cl

onin

g la

rge

(10.

4 to

20

kb)

frag

men

ts.

It c

onta

ins

a po

lylin

ker

with

clo

ning

site

s fo

r B

amH

I, S

alI,

and

Eco

RI;

in E

MB

L4,

the

poly

linke

r is

rev

erse

d. R

ecom

bina

nt p

hage

s co

ntai

ning

DN

Ain

sert

ed in

to t

hese

site

s be

com

e cI

− , ga

m− ,

red− ,

and

int− ,

and

thus

hav

e th

e S

pi−

phen

otyp

e. A

lthou

gh th

e ph

age

is s

aid

to c

arry

cI8

57, w

e be

lieve

that

it d

oes

not c

arry

a cI

ge

ne,

and

it

has

bee

n d

raw

n a

cco

rdin

gly

. T

he

pol

ylin

ker

sequ

ence

is

GG

ATC

TG

GG

TC

GA

CG

GAT

CC

GG

GG

AAT

TC

CC

AG

ATC

C. E

MB

L4’s

full

geno

type

isλs

bhI λ

1 ° b

189

< po

lylin

ker

(Sal

I-E

coR

I) in

t29

ninL

44 c

I857

trp

E p

olyl

inke

r (E

coR

I-S

alI)

> K

H54

chi

C s

rIλ4

° ni

n5 s

rIλ5

° (F

risch

auff

et a

l., 1

983)

.

Current Protocols in Molecular Biology

Sal I Bam HI

Eco RI

Bgl II

Pvu I

WD

M

AB

CE

ZU

GV

TI

HL

K

pL

p RM

p Rp R

EE

MB

L3p tr

pp

L

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

44

Jkb

KH

54ni

n5

WD

MR

z

att

AB

CE

ZU

GV

TI

HL

KP

401

Ea3

1

int

Ea5

9N

c

lO

QS

R

p L

p R

p RMp R

Ep

I

λ+

J

int 2

9N

trpE

b189

chiC

Sal

IB

am H

IE

co R

IS

al I

Bam

HI

Eco

RI

Rz

PN

OQ

SR

chiC

cll

trpE

int2

9

Ngi

t

att

Kpn I

SmaI

Sal I

Bgl IIPvuI

HindIII

Bgl II

HindIII

EcoRIBam HI

Sal I

Sma ISst I

HindIII

p′R

(con

tinue

d)

p′R

(con

tinue

d)p ′

R

p′R

pI

exo

gam

Kpn I

Sal I

Bgl II

Bgl IIBgl II

Bgl IIBgl II

Sma I

Nu1

Nu3

FIFII

lom

xis

Ea22Ea8.5

exobetgamgit

rex Brex A

cro

Nu1

Nu3

FlFll

lom

314194

Ea47

xisEa8.5

Ea22exobetgamKclllssbralgit

rex Arex B

cll

146ren

290

6057

cro

20468

56

221

��

��

��

�

��

nin

L44

1.10.2


Fig

ure

1.1

0.3

λ200

1.

Thi

s ph

age

is u

sed

for c

loni

ng la

rge

(10.

4 to

20

kb) D

NA

frag

men

ts. P

hage

s co

ntai

ning

fore

ign

DN

Abe

com

e cI

− , gam

− , int

− , and

red

− . Sin

ce r

ecom

bina

nts

are

red− a

nd g

am− , t

hey

have

the

Spi

− phe

noty

pe, a

nd fo

rm p

laqu

eson

a s

trai

n ly

soge

nic

for b

acte

rioph

age

P2

(see

UN

ITS

1.9

and

1.1

0 ). T

he v

ecto

r con

tain

s cl

onin

g si

tes

for B

amH

I, E

coR

I, H

indI

II,S

acI,

Xba

I, an

d X

hoI.

The

pol

ylin

ker

sequ

ence

is T

CTA

GA

GC

TC

GA

GG

ATC

CA

AG

CT

TC

AAT

TC

TAG

A. λ

2001

’s fu

ll ge

noty

peis

λsb

hIλ1

° b18

9 Εi

nt(li

nker

) srI

λ3° n

inL4

4 Εs

hndI

II λ4

(bio

) (lin

ker)

δ(s

bhI λ

3-sb

hIλ4

) KH

54 s

rIλ4

° ch

iC n

in5

srI λ

5 ° s

hndI

II λ6 °

(Kar

n et

al.,

198

4).

Bgl II

LR

M

p R

RE

λ200

1bi

oBF

CD

L

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

kb

exo

gam

Nbi

o K

H54

WD

M

att

AB

CE

ZU

GV

TI

HL

K40

1E

a31

int

Ea5

9N

p

p R

RMR

EI

λ+

J

b189 H

in d

IIIE

co R

IX

ba I

Bam

HI

Xho

IS

ac I

Xba

I

Rz

NQ

SR

chiC

cll

bio

N

Kpn I

Sma I

Sal I

Xho I

Bgl II

Sma ISst II

Hin

dIII

Eco

RI

Xba

IB

am H

IX

ho I

Sac

IX

ba l

p bi

oA

Xba lSac IXho I

Bam HIHin dIIIEco RI

Xba I

XbaI

Eco RIHin dIIIBam HI

Xho I

Sac IXba l

p′R

(con

tinue

d)

p′R

p′R

p′R

(con

tinue

d)

l

Kpn I

Sma I

Sal I

Bgl IBgl I

Bgl IIBgl II

Bgl IIBgl II

WD

M

AB

CE

ZU

GV

TI

HL

KJ

OP

nin5

Rz

PO

QS

R

chiC

cl

nin

L44

Nu1

Nu3

FlFll

314

lom

Ea8.5

194

Ea47

Ea22

xis

exobetgamBglllclllssbralgitrex Brex A

crocllren14629057605620468221

Nu1

Nu3

FlFll

lom

intxis

Ea22Ea8.5

exobetgamgit

rex Brex A

git

cro

L

p

p

p

p p

p p p

p

1.10.3


Fig

ure

1.1

0.4

λgt1

0.

Thi

s ph

age

vect

or a

ccep

ts s

mal

l (0

to 5

kb)

DN

A fr

agm

ents

whi

ch a

re in

sert

ed in

to a

n R

I site

in it

s cI

gene

. Ins

ertio

n of

DN

A in

to t

his

site

inac

tivat

es t

he c

I ge

ne a

nd e

nabl

es in

sert

-con

tain

ing

phag

es t

o fo

rm p

laqu

es o

n a

hfl−

host

. Sin

ce in

sert

-con

tain

ing

phag

e fo

rm p

laqu

es v

ery

effic

ient

ly o

n th

is h

ost,

and

sinc

e th

e pl

aque

s fo

rmed

by

inse

rt-c

onta

inin

gph

age

are

usua

lly v

ery

heal

thy,

λgt

10 is

ver

y fr

eque

ntly

use

d to

con

stru

ct li

brar

ies

for

whi

ch o

nly

very

sm

all a

mou

nts

of D

NA

are

avai

labl

e, fo

r ex

ampl

e in

the

cons

truc

tion

of c

DN

A li

brar

ies.

Inse

rt-c

onta

inin

g λg

t10

phag

e ar

e cI

− and

int− b

ut r

ed+ . I

ts fu

llge

noty

pe is

λb5

27 s

rIλ3

° im

m43

4 srI

λ4 °

srI

λ5°

(Huy

nh e

t al.,

198

4).

cll

ren 146 290

57 60 56 204 68 221

Bgl II

Sma ISst II

Bam HI

Bgl IIHpa I

λgt1

0

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

kb

4445

Kpn I

pL

pR

p RM

p RE

pI

Ava IHpa I

Bam HIHpa I

Bcl I

Pvu I

Ava ISst II

Bam HI

Hin dllIBgl II

Sma I

Sma I

Bcl I

Xho I

Sal l

Hin dllIEco RI

Bcl I

imm

43

4

b5

27

cro rex A

rex B

gam

Nu1

W Nu3

FI

FII

DM

Ea22

bet

cll

Rz

exo

clll ssb ral git

ren 146 290 57 60 56 204 68 221

194

Ea47

xis

314

att

Ea8.5

Iom

AB

CE

ZU

GV

TI

HL

KP

401

Ea

31

int

Ea

59

Ncl

OQ

SR

pL

pR

p′ R

p RM

p RE

p I

λ+

K

J

p′ R

(co

nti

nu

ed

)

p′ R

(co

nti

nu

ed

)p

′ R

Hpa IHpa I

Bcl I

Hpa I

Bcl I

Hpa I

Kpn I

Sst IIAva ISst IIHpa I

Ava IBam HI

Hpa IHpa I

Sal l

Bam HIHpa I

Hpa I

Ava I

Bgl IIHpa IHpa I

Bcl IHin dllI

Bcl I

Bcl I

Nu1

W

Nu3

FI

FII

DM

194 314

Iom

AB

CE

ZU

GV

TI

HL

K 4

01J

Rz

SR

gam

Ea22

bet exo

clll ssb ral git

Ea47

xisEa8.5

P

in

tN

Q

K

imm

43

4

O

1.10.4


Fig

ure

1.1

0.5

λgt1

1.

Thi

s ph

age

vect

or c

an a

ccep

t sm

all (

0 to

4.8

kb)

DN

A fr

agm

ents

inse

rted

into

the

Eco

RI s

ite lo

cate

dat

the

end

of th

e la

cZ g

ene.

If th

ese

frag

men

ts c

onta

in a

cod

ing

sequ

ence

in fr

ame

with

the

lacZ

cod

ing

sequ

ence

, the

n th

ein

sert

ed D

NA

s ar

e ex

pres

sed

in p

hage

-infe

cted

cel

ls a

s fu

sion

pro

tein

s. P

laqu

es m

ade

by p

hage

s en

codi

ng la

cZ f

used

infr

ame

to t

he c

odin

g se

quen

ce o

f a

give

n pr

otei

n ca

n be

iden

tifie

d by

the

ir ab

ility

to

reac

t w

ith a

ntis

erum

aga

inst

the

nat

ive

prot

ein.

Rec

ombi

nant

pha

ge a

re c

I− , in

t+ , an

d re

d+ . Rec

ombi

nant

s ca

n fo

rm ly

soge

ns f

rom

whi

ch fu

sion

-pro

tein

pro

duct

ion

can

be in

duce

d by

gro

win

g th

e ly

soge

n at

42 °

C. λ

gt11

’s fu

ll ge

noty

pe is

λla

c5 s

rIλ3

° cI8

57 s

rIλ4

° nin

5 sr

I λ5 °

Sam

100

(You

ngan

d D

avis

, 198

3).

rex A

Bam HI

cI8

57

Bgl II

Bgl I

Sst II

Sma I

Xho I

Bam HI

Sal I

Hin dIII

Bgl IIλgt1

1

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

4445

46

kbKpn I

pL

pR

p′ R

p RM

pI

p Iac

Bam HI

Pvu I

Sst I

Eco RI

lac5

( la

cZ )

cro rex A

rex B

gam

Ea22

bet

cll

Rz

exo

clll ssb

ral git

ren 146 290 57 60 56 204 68 221

194

Ea47

xis

314

att

Ea8.5

Iom

P 4

01E

a3

1

in

tE

a5

9N

clO

QS

R

pL

pR

p′ R

p RMp R

Ep

I

p′ R

(co

nti

nu

ed

)

λ+

K

cI8

57

Sa

m1

00

cro

rex B

K

Ea22

bet

cll

exo

clll ssb

ral git

xis

att

Ea8.5

PE

a31

int

Ea5

9N

clO

lac5

( la

cZ )

Q

Xba ISst I

Bgl II

Sma I

Rz

SR

nin

5 Sa

m1

00

p RE

Pvu I

p′ R

(co

nti

nu

ed

)

Nu1

W

Nu3

FIFII

DM

AB

CE

ZU

GV

TH

LJ

Kpn I

Pvu IPvu I

Pvu I

Sst I

Pvu I

Hin dIII

Hin dIIIHin dIIIHin dIII

Sal I

Pvu I

Bgl II

Hin dIII

IK

Nu1

W

Nu3

FIFII

DM

AB

CE

ZU

GV

TH

LJ

IK

gam

1.10.5


Fig

ure

1.1

0.6

Ch

aro

n 4

a.

Thi

s ph

age

was

use

d to

con

stru

ct m

any

earli

er li

brar

ies

whi

ch a

re s

till b

eing

use

d. It

con

tain

sam

ber

mut

atio

ns in

the

A a

nd B

gen

es, a

nd s

o m

ust b

e pr

opag

ated

on

a ho

st c

onta

inin

g ei

ther

Su1

or

Su3

(se

e U

NIT

1.4

). It

can

acco

mm

odat

e la

rge

(7.1

to 2

0.1

kb)

inse

rted

Eco

RI f

ragm

ents

or

som

ewha

t sm

alle

r (0

to 5

.6 k

b) in

sert

ions

into

its

Xba

Isi

te. R

ecom

bina

nt p

hage

s be

arin

g in

sert

ed E

coR

I fra

gmen

ts a

re B

io− a

nd L

ac− , w

hile

pha

ges

bear

ing

inse

rted

Xba

I fra

gmen

tsar

e B

io+ a

nd L

ac+ . I

ts g

enot

ype

is λ

Aam

32 B

amI l

ac5

bio2

56 ∆

KH

54 s

rIλ4

° ni

n5 Q

SR

80 (

Bla

ttner

et a

l., 1

977;

Will

iam

s an

dB

lattn

er, 1

979;

deW

et e

t al.,

198

0). T

he Q

SR

80 s

ubst

itutio

n co

ntai

ns a

sho

rt s

tret

ch o

f DN

A fr

om w

ild-t

ype

λ.Bam HI

Hin dIII

Bgl IICha

ron

4a

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

kb

4445

46

Kpn I

pL

p RM

pR

p bio

Ap

Iac

p′ R

(co

nti

nu

ed

)

Bam H

Pvu I

Sst IEco RIla

c5 (

lacZ

)

crorex A

rex B

gam

Nu1

W

Nu3

FI

FII

DM

Ea22

bet

cll

Rz

exo

clll ssb

ral git

ren 146 290 57 60 56 204 68 221

194

Ea47

xis

314

att

Ea8.5

Iom

AB

CE

ZU

GV

TI

HL

KP

401

Ea

31

int

Ea

59

N

cl

OQ

SR

pL

pR

p′ R

p RMp R

Ep

I

p′ R

(co

nti

nu

ed

)

λ+

K

J

cro cll

PO

lac5

( la

cZ )

Pvu I

Xba ISst I

nin

5

Aa

m 3

2B

am

1

bio

256

K

H5

4

QS

R80

N

bio

256

QS

R80

Aa

m 3

2B

am

1

Bgl II

Bam HIKpn I

p RE

p b

ioB

FC

Dp

′ R

Kpn I

Pvu IPvu I

Pvu I

Sst IEco RI

Pvu IHin dIII

Bgl II

Eco RI

Bgl IIPvu I

Bgl IIBgl II

Bgl II

Bam HISst I

Hin dIII

Bgl II

Sst IBam HI

Bgl II

Nu1

W

Nu3

FI

FII

DM

AB

CE

ZU

GV

TI

HL

KJ

Ea

31

Ea

59

1.10.6


Fig

ure

1.1

0.7

Ch

aro

n 4

0.

Cha

ron

40 i

s a

repl

acem

ent

vect

or t

hat

is u

sefu

l fo

r cl

onin

g ve

ry l

arge

(up

to

24 k

b) D

NA

frag

men

ts. T

he “p

olys

tuffe

r” is

flan

ked

by p

olyl

inke

rs c

onta

inin

g 16

rest

rictio

n si

tes

incl

udin

g se

vera

l site

s th

at a

re n

ot a

vaila

ble

in o

ther

vec

tors

. The

se s

ites

are

not p

rese

nt in

the

λ ar

ms.

The

pol

ystu

ffer i

s co

mpo

sed

of re

peat

s of

a 2

35-b

p D

NA

frag

men

t;it

can

be r

educ

ed to

sm

all f

ragm

ents

by

dige

stio

n w

ith N

aeI.

The

se s

mal

l pie

ces

are

easi

ly r

emov

ed b

y po

lyet

hyle

ne g

lyco

lpr

ecip

itatio

n. T

he re

com

bina

nts

reta

in g

am, w

hich

enc

odes

an

inhi

bito

r of t

he re

cBC

nuc

leas

e, a

nd th

us a

re s

tabl

y pr

opag

ated

even

if r

epea

ted

sequ

ence

s ar

e pr

esen

t in

the

inse

rt. E

ven

grea

ter

stab

ility

can

be

achi

eved

by

grow

th o

f a v

ecto

r on

a r

ecA

E. c

oli.

Cha

ron

40A

is id

entic

al to

Cha

ron

40, e

xcep

t tha

t it c

onta

ins

the

Aam

32 a

nd B

amH

1 m

utat

ions

(D

unn

and

Bla

ttner

,19

87).

Bgl II

Pvu I

Bgl IIPvu Icro

Rz

SR

cll

git

PN

OQ

~80

copi

es (l

ac o

pera

tor

+ a

deno

DN

A) (

235b

p)( p

olys

tuffe

r)

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

4445

46

kb

Sma IXba ISal l

Eco RlSac IKpn l

Nae I

Avr IISpe IXho lApa I

Bam HISfi l

Hin dIIINot I

Xma lII

Xma lII

Xma lII

Nae l

~80

x

Nu1

W

Nu3

FIFII

DM

AB

CE

ZU

GV

TI

HL

KJ

p′R

(con

tinue

d)

p Lp R

M

p Rp′

R

p RE

Cha

ron

40

Kpn lSac I

Eco RI

Nae I

~80

copi

es la

c op

erat

or +

ade

no D

NA

(23

5bp)

KH

54ni

n5W

.L.

113

crorex Arex B

gam

Nu1

W

Nu3

FI

FII

DM

Ea22

bet

cll

Rz

exo

clll ssb ral git

ren14629057605620468221

194

Ea47

xis

314

att

Ea8.5

Iom

AB

CE

ZU

GV

TI

HL

KP

401

Ea3

1

int

Ea5

9N

clO

QS

R

p L

p Rp′

R

p RMp R

Ep

I

p′R

(con

tinue

d)

λ+

K

J

Sal lXba ISma I

Xho lSpe IAvr II

Sfi lBam HI

Apa I

Xma lIINot I

Hin dIIIBgl IIBgl IIBgl II

1.10.7


Fig

ure

1.1

0.8

λZA

P.

λZ

AP

car

ries

pBlu

escr

ipt S

K ( −

), w

hich

is e

xcis

ed in

viv

o up

on in

fect

ion

with

f1 o

r M13

hel

per p

hage

s.In

sert

s ar

e cl

oned

into

λZ

AP

with

in B

lues

crip

t se

quen

ces.

Spe

cific

ally

, in

sert

s ar

e cl

oned

with

in a

pol

ylin

ker

loca

ted

with

inla

cZ. A

s w

ith λ

gt11

, a fu

sion

pro

tein

may

be

expr

esse

d if

the

inse

rt D

NA

is in

fram

e w

ith th

e la

cZ s

eque

nce;

thus

, lib

rarie

sm

ade

in th

is v

ecto

r can

be

scre

ened

with

ant

ibod

ies.

In λ

ZA

P, T

7 an

d T

3 pr

omot

ers

flank

the

inse

rts,

whi

ch a

llow

s R

NA

pro

bes

to b

e ea

sily

obt

aine

d. p

Blu

escr

ipt

M13

(−)

, th

e ex

cise

d pl

asm

id (

show

n on

the

rig

ht),

is

norm

ally

pro

paga

ted

as a

doub

le-s

tran

ded

circ

ular

DN

A,

but

infe

ctio

n w

ith a

hel

per

phag

e en

able

s th

e pl

asm

id t

o be

pro

paga

ted

as s

ingl

e-st

rand

edD

NA

. The

se p

rope

rtie

s fa

cilit

ate

sequ

enci

ng o

f th

e in

sert

, si

te-d

irect

ed m

utag

enes

is,

and

the

cons

truc

tion

of u

nidi

rect

iona

lde

letio

ns. D

NA

frag

men

ts u

p to

10

kb c

an b

e in

sert

ed. W

ithin

the

poly

linke

r, un

ique

Xho

I, E

coR

I, S

peI,

Xba

I, N

otI,

and

Sac

Icl

onin

g si

tes

are

avai

labl

e. λ

ZA

P/R

is s

how

n in

the

figur

e. λ

ZA

P/L

is id

entic

al to

λZ

AP

/R e

xcep

t tha

t the

pol

ylin

ker i

s in

vert

ed.

λZA

P v

ecto

rs a

re a

vaila

ble

from

Str

atag

ene.

T3

T7

SacI Not I XbaI SpeI

XhoI

BamHI*

EcoRI

SalI*

KpnI*

SmaI*

HindIII

SacINotIXbaISpeI

BamHI

SmaIEcoRI

SalIXhoIKpnI

T3

T7

Co

l E

Io

riT

am

p

lacZ

′ I

c185

7

Blu

escr

ipt

M13

–29

50bp

M13 o

ri

a

mp

ori

lacIP

lac

lac

Z′

Cfr

101

212

5C

su I

2130

Pvu

I 241

2

Sca

I 252

1

Xm

nI 2

640

Ssp

I 284

5N

aeI–

118

I

Ssp

I 439

456

T3

pro

mot

er

T7

pro

mot

er

AfI

II 11

52

Kpn

I

Apa

I

Xho

I

Sal

I

Cla

I

Hin

dIII

Eco

RV

Eco

RI

Pst

I

Sm

aI

Bam

HI

Spe

I

Xba

I

Not

I

Eag

I

Bst

XI

Sac

II

Sac

I

T7

T3

Pvu

II 97

2

Pvu

I 498

Pvu

II 52

7

crorex A

rex B

gam

Nu1

W

Nu3

FI

FII

DM

Ea22

bet

cll

Rz

exo

clll ssb

ral git

ren 146 290 57 60 56 204 68 221

194

Ea47

xis

314

att

Ea8.5

Iom

AB

CE

ZG

VT

IH

LK

P 4

01E

a3

1in

tE

a5

9N

clO

QS

R

pL

pR

p′ R

p RMp R

Ep

I

p′ R

(co

nti

nu

ed

)

λ+

K

J

Sam

100

chi A

131

HindIII* HindlII

Q

rex A rex B

gam

Ea22

bet exo

clll ssb

ral git

xis

att

Ea8.5

int

N

cl

K

BglII

PvuI

BglII BglI

cro

Nu1

W

Nu3

FI

FII

DM

cll

Rz

AB

CE

ZU

GV

TI

HL

KP

OS

R

p′ R

(co

nti

nu

ed

)

p L

p R

M

p R

p′ R

p R

E

λZA

P/R

01

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

40

JK

b

chi A

131

Co

l E

Io

riT

am

p

lacZ

′ I

c185

7S

am 1

00

KpnI

KpnI

PvuI

BamI

SmaI

SalI SalI

BamI BglII PvuI

HindIII

SmaI

p am

pp la

c

pI

PvuI

HindIII HindIII

BglII

HindIII

nin

5

U

1.10.8

THE COSMID, A USEFUL LAMBDA-DERIVED PLASMID VECTOR

Cosmids were developed to allow cloningof large pieces of DNA. How they work isdiagrammed in Figure 1.10.9. Cosmids containa selectable marker, a plasmid origin of repli-cation, a site into which DNA can be inserted,and a cos site from phage λ. The vector is cutwith a restriction enzyme and mixed withpieces of DNA to be cloned. DNA ligase joinscut vector and insert fragments into concate-meric molecules. The ligation mixture is thenmixed with a packaging extract (see UNIT 1.11)containing the proteins necessary to package

naked phage DNA into phage heads. Whenevertwo cos sites are present on a concatemer andseparated by 40,000 to 50,000 nucleotides,they will be cut and packaged into phage heads.The cosmid-containing phages are infectious,they inject DNA into cells, but the DNA isplasmid DNA: the phage adsorbs to the hostand the cosmid DNA is injected into the cell,which circularizes due to its sticky ends. Theannealed ends are then covalently joined bythe host’s ligase, and the resulting large cir-cular molecule replicates as a plasmid (seemap, Fig. 1.10.10).

Cosmid vectors within cells replicate using

Figure 1.10.9 Cloning into cosmids.

oricosampr

digest

eukaryotic DNA

package intophage

ampr

ori

cos

digest

restrictionsite

ligate

cloning into cosmid vectors

infect E. coli

select transformants


1.10.9


their pBR322 origins. Intracellular cosmidssometimes rearrange DNA inserted into them,perhaps because the time it takes pBR322-de-pendent replication to replicate the 50,000-bpcosmid is almost as long as the generation timeof E. coli, so that cosmids which have deletedsections of DNA have a growth advantage onantibiotic-containing medium. Although thisproblem can be ameliorated by propagating thecosmids in cells that are less prone to rearrangevector DNA (UNIT 1.4), another approach maywell become more popular. Cosmid vectors(called lorist vectors) have recently been devel-oped that are said to circumvent this problemby using the λ ori and O and P proteins toreplicate (see, for example, Gibson et al., 1987).It takes only a few minutes for these vectors toreplicate inside cells, and insertions in them aresaid to be more stable.

LITERATURE CITEDBanuett, F., Hoyt, A.M., McFarlane, L., Echols, H.,

And Herskowitz, I. 1986. hflB, a new Es-cherichia coli locus regulating lysogeny and thelevel of bacteriophage lambda cII protein. J. Mol.Biol. 187:213-224.

Blattner, F.R., Williams, B.G., Blechl, A.E., Dennis-ton-Thompson, K., Faber, H.E., Furlong, L.-A.,Grunwald, D.J., Kiefer, D.O., Moore, D.D.,Schumm, J.W., Sheldon, E.O., and Smithies, O.1977. Charon phages: Safer derivatives of bacte-riophage lambda for DNA cloning. Science196:161-169.

deWet, J.R., Daniels, D.L., Schroeder, J.L., Wil-liams, B.G., Denniston-Thompson, K., Moore,D.D., and Blattner, F.R. 1980. Restriction mapsfor twenty-one Charon vector phages. J. Virol.33:401-410.

Dunn, I.S. and Blattner, F.R. 1987. Charon 36 and40: Multi-enzyme, high capacity, recombinationdeficient replacement vectors with polylinkersand polystuffers. Nucl. Acids Res. 15:2677-2698.

Frischauf, A.-M., Lehrach, H., Polstka, A., and Mur-ray, N.M. 1983. Lambda replacement vectorscarrying polylinker sequences. J. Mol. Biol.170:827-842.

Gibson, T.J., Coulson, A.R., Sulston, J.E., and Little,P.F.R. 1987. Lorist 2, a cosmid with transcrip-tional terminators insulating vector genes frominterference by promoters within the insert: Ef-fect on DNA yield and cloned insert frequency.Gene 53:275-281.

Hendrix, R., Roberts, J., Stahl, F., and Weisberg, R.1983. Lambda II. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y.

pJB8~5400bp

5.4/0

5.0

4.0

3.0

2.0

1.0

cos

ampr

ClaIIIHindIII

EcoRI BamHI EcoRI

PstI

AvaISalI

Figure 1.10.10 pJB8 is a commonly used cosmid vector. It contains a cos site, an ampicillinresistance gene, and a pMB1 replicator, so it can be amplified with chloramphenicol. In a typicalapplication, libraries are constructed by insertion of random Sau3a partial-digestion fragments ofDNA into its BamHI site. The inserts can be excised by cleaving the plasmid at its flanking EcoRIsites (Ish-Horowitz and Burke, 1981).


1.10.10

Lambda as aCloning Vector

Hoyt, M.A., Knight, D.M., Das, A., Miller, H.I., andEchols, H. 1982. Control of phage lambda devel-opment by stability and synthesis of cII protein:Role of the viral cIII and host hflA, himA, andhimD genes. Cell 31:565-573.

Huynh, T.V., Young, R.A., and Davis, R.W. 1984.Constructing and screening cDNA libraries inλgt10 and λgt11. In DNA Cloning Techniques:A Practical Approach (D. Glover, ed.) pp. 49-78.IRL Press, Oxford.

Ish-Horowitz, D. and Burke, J.F. 1981. Rapid andefficient cosmid cloning. Nucl. Acids Res.9:2989-2998.

Karn, J., Matthes, H.W.P., Gait, M.T., and Brenner,S. 1984. A new selection cloning vector, λ2001,with sites for XbaI, BamHI, HindIII, EcoRI, SstI,and XhoI. Gene 32:217-224.

Williams, B.G. and Blattner, F.R. 1979. Construc-tion and characterization of the hybrid bacterio-phage lambda Charon vectors for DNA cloning.J. Virol. 29:555-575.

Young, R.A. and Davis, R.W. 1983. Efficient isola-tion of genes by using antibody probes. Proc.Natl. Acad. Sci. U.S.A. 80:1194-1198.

Zissler, J., Signer, E., and Schaefer, F. 1971. The roleof recombination in growth of bacteriophagelambda. In The Bacteriophage Lambda (A.D.Hershey, ed.) pp. 455-475. Cold Spring HarborLaboratory, Cold Spring Harbor, NY.

KEY REFERENCEMurray, N.E. 1983. Phage Lambda and Molecular

Cloning. In Lambda II, (R.W. Hendrix, J.Roberts, F. Stahl, and R. Weisberg, eds.) pp.395-432. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY.

Provides a thorough introduction to the biology ofcloning vectors in common use before 1983.


Nina IrwinHarvard UniversityCambridge, Massachusetts


1.10.11


UNIT 1.11 Plating Lambda Phage to Generate PlaquesBASIC

PROTOCOLISOLATING A SINGLE PLAQUE BY TITERING SERIAL DILUTIONS

This procedure is used to isolate pure populations of phage from a single plaque andprovide the titer of the phage stock. Serial dilutions are made of a phage lysate. In separatetubes, aliquots of each dilution are mixed with E. coli. Phage are allowed to adsorb to thecells and the cell/phage mixture is then heated to 37°C, causing the phage to inject theirDNA into the cells. Top agar is added to each tube, and the mixture is poured onto richplates, which are incubated at 37°C until plaques appear. Each plaque contains phagesderived from a single infecting phage.

Materials

Lambda broth (UNIT 1.1)0.2% maltose10 mM MgSO4

Lambda top agar (UNIT 1.1)Suspension medium (SM)Fresh lambda plates (UNIT 1.1), prewarmed to 37°C

Microwave oven or boiling water bath45° to 50°C water bath8 × 80–mm tubes37°C water bath or heat blockCapillary tubes or toothpicks

NOTE: All materials coming into contact with E. coli must be sterile.

1. Grow a culture of E. coli to saturation in lambda broth + 0.2% maltose + 10 mMMgSO4.

Growth in maltose induces production in E. coli of the λ receptor (lamB protein), which isnecessary for maltose transport. Mg++ ions also aid phage adsorbtion.

2. Melt top agar in microwave oven set to defrost setting, or in boiling water bath for15 min. After agar is melted, let bottle cool at room temperature for 5 min, then placebottle of melted agar in 45° to 50°C water bath.

The cap to the bottle of top agar must be loose before putting it in microwave! Watch themicrowave oven to make sure that the contents of the bottle of top agar do not boil over. Ifagar boils over, carefully take the bottle out and swirl it around to see if there are anyunmelted flecks of agar. If there are, reinsert into oven and microwave longer, inspectingoccasionally, until the agar is completely melted.

Be sure top agar is left in the water bath enough time to cool to 45° to 50°C. Cells will bekilled by even brief exposure to agar that is hotter than 65°C.

3. Add 0.3 ml of the E. coli culture to five 8 × 80–mm tubes.

4. Make serial dilutions of the phage lysate in SM (see UNIT 1.3 for serial dilutions).

Dilution factors of 100-fold are usually used. Label the dilution tubes so that they do notget mixed up.

5. Add 0.1 ml of the first dilution to one tube of E. coli, 0.1 ml of the next dilution tothe next tube, etc. Label the tubes of E. coli/phage mixture, so that they do not getmixed up. Incubate tubes at room temperature for 20 min.

The phage adsorb to the E. coli during this step.

6. Move tubes to a 37°C water bath or heat block for 10 min. While tubes are in

Contributed by Karen Lech and Roger BrentCurrent Protocols in Molecular Biology (1990) 1.11.1-1.11.4Copyright © 2000 by John Wiley & Sons, Inc.Supplement 13

1.11.1

Plating LambdaPhage to Generate

Plaques

water bath, label 5 fresh, prewarmed lambda plates to correspond with the labels onthe dilution tubes.

During this step, the phage inject their DNA into the cells.

Plates should be fresh, but not so fresh that they are wet on their surfaces, nor so wet thatthey will exude moisture and cause the lawn of cells in top agar to slide away.

7. Remove tubes from the water bath. Add 2.5 ml top agar to one tube, vortex lightly tomix, and pour the contents of tube onto a plate. Spread agar over the entire surfaceof the plate by tilting it gently.

8. Place the plates in a 37°C incubator. Plaques of lambda-derived phages will appearafter 6 to 8 hr, but will be easier to score, count, and pick if left for 12 hr.

9. From one of the dilution plates that is not too crowded with plaques, pick a singleplaque with a sterile capillary tube or toothpick. To save the plaque for future use (forexample, to make a plate stock), cut out a plug of agar containing the plaque with acapillary tube, and blow the plug into a tube containing 1 ml of SM (or place the tipof the toothpick in the liquid and agitate gently). If desired, count the number ofplaques on one of the dilution plates and use this number to compute the number ofviable phage in the starting stock.

Background Information

Titration of bacteriophage, and isolation of phage from single plaques, was first describedby d’Herelle in 1920. The best general background to bacteriophage growth protocols isprobably found in Stent (1971).

Genes encoding the tail proteins of most lambda-derived cloning vectors come from phageλ. Vectors with these tail proteins, said to be “hλ” (for host range of λ), adsorb to the celllamB protein, which is involved in maltose uptake. These vectors make plaques with sharpboundaries. Some vectors have tail proteins derived from phage 80 and are said to be“h80.” These vectors adsorb to the host tonA protein, which is involved in ion transport.They make slightly larger plaques with fuzzier borders than those made by hλ phages.Stocks of h80 phages usually have a higher titer than stocks of corresponding hλ phages.

Troubleshooting

It is sometimes helpful to include two other control plates in the procedure. One is a platethat contains a lawn made from a separate tube of the E. coli culture that has not beeninfected with phage. The other is a plate that contains top agar that did not contain anycells. These two controls provide benchmarks for growth of the lawn, as it becomes denserduring the time in the incubator. If the lawn appears crinkled, then the top agar/E.coli/phage mixture was probably too cold by the time it was poured onto the plate. If thisoccurs, try warming the plates up to 37°C before pouring the lawn onto them, or pouringthe lawns more quickly after the top agar is added to the E. coli/phage mixture. If the topagar layer floats off the plate, then the plates were too wet. If this occurs, use dryer plates.

Time Considerations

It takes from 6 to 8 hr of incubation at 37°C before plaques appear, and is often 12 to 14hr before differences in morphology can be reliably distinguished.


1.11.2


BASICPROTOCOL

ISOLATING SINGLE PLAQUES BY STREAKING ONA LAWN OF CELLS

Phages are streaked for single plaques on a plate containing a prepoured lawn of E. coli.This procedure is easier than titering by serial dilutions, and is recommended if only afew isolated plaques are needed.

Materials

LB medium (UNIT 1.1)Lambda top agar (UNIT 1.1)Rich plate (UNIT 1.1), prewarmed to 37°C32-G platinum wire loop or sterile 11⁄2 × 1⁄8 in. strips of paper

1. Grow a lambda-sensitive strain of E. coli to saturation in 5 ml LB medium.

2. Add 0.2 ml of the saturated culture to 2 ml of melted top agar (cooled to about 45°C;see step 2, p. 1.11.1), and pour evenly over the top of a prewarmed, rich plate.

In the recipe for top agar (UNIT 1.1), agar can be replaced with 6 g agarose.

3. After top agar has hardened, cool plates by placing in refrigerator ≥15 min.

4. Spot 100 µl of λ stock culture (usually around 108 phage/ml) on the corner of theplate.

5. Using the techniques described in UNIT 1.3 to streak out single bacterial colonies, lightlystreak out the phage using a thin wire loop or the edge of a sterile 11⁄2 × 1⁄8 in. pieceof paper.

Paper should be cut into strips and sterilized by autoclaving (dry) in screw-cap vials.

BASICPROTOCOL

PHAGE TRANSFECTION AND IN VITRO PACKAGING

Construction of a library with lambda-derived cloning vectors results in a population ofphage DNA molecules. In order for these molecules to be replicated, they must beintroduced into cells so that they can grow as phage. Phage DNA is typically introducedinto cells either by infection after packaging into phage particles in vitro or, much lessfrequently, by transfection, that is, transformation of phage DNA that has been circular-ized by treatment with DNA ligase into competent cells.

In vitro packaging uses lysates of phage-infected E. coli called packaging extracts. Theselysates contain empty phage heads, unattached phage tails, and the phage-encoded proteinsrequired for DNA packaging (see UNIT 1.9). If ATP is present, then concatemeric phage DNAmixed with the extract is cut at one cos site by the terminase (probably a complex of the Aand Nul proteins) and loaded into the phage head by an unknown mechanism. DNA continuesto be loaded into the phage head until the terminase encounters and cuts the next cos site onthe molecule. The phage tails then attach themselves to the filled heads.

In the early 1980s, production of high-efficiency packaging extracts was considered oneof the biggest inconveniences in library construction. To avoid the work involved, mostresearchers now use frozen packaging extracts purchased from commercial suppliers(see Enquist and Sternberg, 1979). Commercial extracts typically yield 2 × 108 to 2 ×109 plaque-forming particles per µg of concatemerized phage DNA. Phage DNA canalso be introduced into E. coli by transformation of competent cells. In this procedure,linear or circular phage DNA is mixed with competent E. coli, and the mixture of cellsand DNA is treated as in UNIT 1.8. After the heat shock step, the calcium-treated cellsare mixed with E. coli that have been grown in lambda broth and maltose asdescribed in first basic protocol. Molten top agar is then added, the mixture is poured


1.11.3

Plating LambdaPhage to Generate

Plaques

onto a lambda plate, allowed to solidify, and incubated at 37°C until plaques appear. Thisprocedure typically yields 104 plaques/µg of phage DNA.


Suspension medium (SM), per liter5.8 g NaCl2 g MgSO4⋅7H2O50 ml 1 M Tris⋅Cl, pH 7.50.01% gelatin (Difco)

LITERATURE CITEDEnquist, L. and Sternberg, N. 1979. Packaging of bacteriophage λ in vitro. Meth. Enzymol. 68:281-298.

Stent, G.S. 1971. Molecular Genetics: An Introductory Narrative, W.H. Freeman, NY.



1.11.4


UNIT 1.12 Growing Lambda-Derived VectorsIt is often necessary to grow large quantities of lambda-derived vectors, so that DNA canbe made from them (see UNIT 1.14). The following basic protocol tells how to make a phagestock by plate lysis, while the alternate protocol tells how to make a liquid lysate. Storageof phage lysates is described in the support protocol.

BASICPROTOCOL

MAKING A STOCK OF PHAGE BY PLATE LYSIS

Phage from a single plaque are mixed with cells and top agar and poured onto a plate.Although there are initially more cells than phage in the lawn, the phage increase theirnumber more rapidly than the cells, and eventually lyse most cells on the plate. The topagar is then scraped off the plate and the phage in it are extracted and saved.

Materials

Fresh lambda top agar (UNIT 1.1)Fresh lambda plates (UNIT 1.1), prewarmed to 37°CSuspension medium (SM; UNIT 1.11)Chloroform

45° to 50°C water bathCapillary tube or toothpickBeckman JA-21 rotor or equivalent


1. Dilute a fresh overnight culture of a lambda-sensitive strain of E. coli 50-fold andgrow in culture tube on a roller drum at 37°C.

2. Melt 100-ml bottle of fresh lambda top agar by heating in microwave oven set todefrost. Place in 45° to 50°C water bath.

3. When cell density reaches 2 to 3 × 108/ml (OD600 = 0.4), place 0.75 to 1 ml of cellsinto a test tube. Pick a single fresh plaque with capillary tube or toothpick, blow theplug into cells, and vortex lightly for 10 sec.

4. Add 7.5 ml top agar, vortex gently, and pour equal amounts of the mixture onto threeprewarmed fresh lambda plates.

If agar in the top agar and plate recipes is replaced by agarose, the extractable DNA willbe an acceptable substrate for restriction enzymes and ligases.

5. Incubate plates at 37°C, typically for 4 to 6 hr, until plaques are clearly visible andwhen 90% to 100% of the lawn is lysed.

6. Pipet 3 ml SM onto plate. Using clean microscope slide, scrape top agar from all 3plates into a small centrifuge tube (e.g., a 15-ml screw-top tube for the BeckmanJA-21 rotor).

7. Add 3 drops chloroform. Vortex vigorously for 10 sec.

8. Leave at room temperature 10 min.

As an alternative to steps 6 through 8, 3 ml SM and 3 drops chloroform can be dribbledonto the plate, the plate left overnight at 4°C, and the liquid decanted and centrifuged asbelow.

9. Centrifuge 10 min at 10,000 rpm in JA-21 rotor (11,400 × g), 4°C.

10. Gently decant and save supernatant.

Contributed by Karen LechCurrent Protocols in Molecular Biology (1990) 1.12.1-1.12.3Copyright © 2000 by John Wiley & Sons, Inc.Supplement 13

1.12.1

Growing Lambda-Derived Vectors

ALTERNATEPROTOCOL

MAKING A LIQUID LYSATE

Host bacteria grown to saturation are infected with 105 to 108 phage/ml. Following phageadsorption, the infection mixture is diluted into a rich medium and shaken vigorously untilcell lysis. Any remaining viable cells are lysed with chloroform, and cell debris is removedwith a low-speed spin.


LB medium (UNIT 1.1)Lambda dilution buffer10 mM MgCl2/10 mM CaCl2

NZC medium (UNIT 1.1)Beckman JA-20 rotor or equivalent

1. Grow an overnight culture of a lambda-sensitive strain of E. coli in LB medium at 37°C.

Lambda-sensitive strains will support lytic growth. This can be tested by spotting 10 �l ofa lambda lysate onto a lawn of bacteria. If the strain is lambda-sensitive, a plaque willform where the phage were spotted.

2. Using a sterile toothpick or capillary tube, pick a single plaque (see step 3 of basicprotocol), blow it into a tube that contains 0.4 ml lambda dilution buffer, and placetube at 4°C for 2 hr to allow phage to elute.

Alternatively, 105 to 108 phage from a liquid lysate or plate stock can be used.

3. Combine 0.1 ml eluted phage with 0.1 ml of saturated culture and 0.1 ml of 10 mMMgCl2/10 mM CaCl2 solution and incubate 15 min in a 37°C water bath.

Incubating with Mg++ and Ca++ allows the phage to adsorb to the bacteria.

4. Transfer this solution to 50 ml of NZC medium and shake vigorously at 37°C untillysis occurs (usually between 6 and 8 hr).

Good aeration is important for high yields.

5. The culture should be checked frequently after 6 hr, and harvested immediately uponclearing.

6. Add a few drops of chloroform to lyse any remaining cells, transfer the solution toCo-rex or Nalgene tubes (being careful to leave the chloroform behind), and spin 10min at 10,000 rpm (12,100 × g), 4°C, to pellet the cell debris.

7. Save as much of the lysate as desired. Transfer to a screw-cap tube, add a few dropsof chloroform, vortex briefly, and store at 4°C.

The titer of the phage should be determined as described in UNIT 1.11.

SUPPORTPROTOCOL

STORING PHAGE LYSATES

Phage stocks should be stored in SM (UNIT 1.11) plus a few drops of chloroform at 4°C.Screw-cap glass tubes with rubber-lined or teflon caps, capable of holding at least 1 ml, aregenerally used, although disposable plastic tubes work well. Lambda titers drop over a periodof several years. Stocks should be dated and checked occasionally to determine phage titer.Addition of 0.1% gelatin to the SM slows down the rate at which phage titers drop whenstored under these conditions. Lysates can also be stored frozen in 15% glycerol.


Lambda dilution buffer20 mM Tris⋅Cl, pH 820 mM MgCl2


1.12.2


COMMENTARY

Background InformationPlate lysis is typically used to make small

phage stocks, which are then used as interme-diates for making larger stocks for preparationof phage DNA. However, if agar is replaced byagarose in the plate recipe and in the top agar(see UNIT 1.1), then DNA extracted from phagein the lysate is an acceptable substrate for re-striction enzymes and ligases.

Liquid lysates generally give slightly loweryields than plate lysates. Less work is required,however, especially when large quantities ofphage are needed. This fact, taken together withthe fact that there is no contaminating agar inthe lysate, makes this method preferable forisolating phage for DNA preps. In the liquidlysis procedure, cells are diluted into NZCmedium after phage adsorption. This is a richmedium that contains amino acids but no glu-cose or other sugars. In cells grown in thismedium, sugar receptors like LamB protein arefractionally induced (so phage can adsorb to thecell), but cells are not covered with so manyreceptors that adsorption to their debris de-pletes the yield of phage from the lysate. Inaddition, cells grown on this medium seem tocause infecting phage to grow lytically, whichhelps to obtain high titers of turbid phages.

Critical ParametersLB medium and top agar can replace lambda

plates and top agar for the plate lysis method,but the phage yield is usually lower. It is impor-tant that the plates be freshly poured, and thatthe top agar be freshly melted. The plates donot even have to be dry; a loose lawn is noproblem as long as the plates are not tilted. Ifthe yield is less than expected, it may be becausethe plates were not fresh, or because the par-ticular phage used does not give a burst as largeas that given by wild-type lambda. The size ofsingle plaques made by the phage sometimesgives a clue that the second problem obtains. Ifplaques made by the phage in question aresmaller than those made by closely relatedphages, it is often worthwhile to reduce thenumber of starting cells, or to increase thestarting number of phage by combining severalplaques, and to incubate the plates longer until

the lawn is completely lysed.In the liquid lysis method, good aeration is

essential for high yields. Flasks should be nomore than 1⁄5 full. Cultures should be watchedcarefully as they approach lysis and harvestedsoon after lysis is complete to prevent releasedphage from reinfecting cell debris. Lysis usu-ally takes between 6 and 8 hr. If there is no signof lysis after 8 hr, there probably will not beany, and one can either go on to add the chlo-roform and complete the procedure, or abandonhope and start over.

Anticipated ResultsThe plate lysis procedure yields ∼10 ml of

a phage stock containing 1010 to 1011 phage/ml. The liquid lysis procedure usually yields astock with 5 × 109 to 3 × 1010 phage/ml.

Time ConsiderationsThe plate lysate procedure usually takes about

8 hr. The liquid lysate procedure takes 2 hr to elutethe phage from the plaque, and 6 to 9 hr betweenthe time the cells are infected and the time thelysate is harvested. The time required to elute thephage is very flexible. Two hours will ensure thatmore than 90% of the phages are eluted from theplaque; shorter elution times allow less phage tobe eluted. Elution times as short as 30 min gen-erally work but do not always give good lysis.Phage may be eluted for longer periods of time;overnight is often convenient. Once the host cellsare infected, it takes ∼6 to 9 hr for lysis andpreparation of the lysate.

Key Reference


Origin of this liquid lysate procedure.

Contributed by Karen LechMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts


1.12.3


UNIT 1.12 Growing Lambda-Derived VectorsIt is often necessary to grow large quantities of lambda-derived vectors, so that DNA canbe made from them (see UNIT 1.14). The following basic protocol tells how to make a phagestock by plate lysis, while the alternate protocol tells how to make a liquid lysate. Storageof phage lysates is described in the support protocol.

BASICPROTOCOL

MAKING A STOCK OF PHAGE BY PLATE LYSIS

Phage from a single plaque are mixed with cells and top agar and poured onto a plate.Although there are initially more cells than phage in the lawn, the phage increase theirnumber more rapidly than the cells, and eventually lyse most cells on the plate. The topagar is then scraped off the plate and the phage in it are extracted and saved.

Materials

Fresh lambda top agar (UNIT 1.1)Fresh lambda plates (UNIT 1.1), prewarmed to 37°CSuspension medium (SM; UNIT 1.11)Chloroform

45° to 50°C water bathCapillary tube or toothpickBeckman JA-21 rotor or equivalent


1. Dilute a fresh overnight culture of a lambda-sensitive strain of E. coli 50-fold andgrow in culture tube on a roller drum at 37°C.

2. Melt 100-ml bottle of fresh lambda top agar by heating in microwave oven set todefrost. Place in 45° to 50°C water bath.

3. When cell density reaches 2 to 3 × 108/ml (OD600 = 0.4), place 0.75 to 1 ml of cellsinto a test tube. Pick a single fresh plaque with capillary tube or toothpick, blow theplug into cells, and vortex lightly for 10 sec.

4. Add 7.5 ml top agar, vortex gently, and pour equal amounts of the mixture onto threeprewarmed fresh lambda plates.

If agar in the top agar and plate recipes is replaced by agarose, the extractable DNA willbe an acceptable substrate for restriction enzymes and ligases.

5. Incubate plates at 37°C, typically for 4 to 6 hr, until plaques are clearly visible andwhen 90% to 100% of the lawn is lysed.

6. Pipet 3 ml SM onto plate. Using clean microscope slide, scrape top agar from all 3plates into a small centrifuge tube (e.g., a 15-ml screw-top tube for the BeckmanJA-21 rotor).

7. Add 3 drops chloroform. Vortex vigorously for 10 sec.

8. Leave at room temperature 10 min.

As an alternative to steps 6 through 8, 3 ml SM and 3 drops chloroform can be dribbledonto the plate, the plate left overnight at 4°C, and the liquid decanted and centrifuged asbelow.

9. Centrifuge 10 min at 10,000 rpm in JA-21 rotor (11,400 × g), 4°C.

10. Gently decant and save supernatant.

Contributed by Karen LechCurrent Protocols in Molecular Biology (1990) 1.12.1-1.12.3Copyright © 2000 by John Wiley & Sons, Inc.Supplement 13

1.12.1


ALTERNATEPROTOCOL

MAKING A LIQUID LYSATE

Host bacteria grown to saturation are infected with 105 to 108 phage/ml. Following phageadsorption, the infection mixture is diluted into a rich medium and shaken vigorously untilcell lysis. Any remaining viable cells are lysed with chloroform, and cell debris is removedwith a low-speed spin.


LB medium (UNIT 1.1)Lambda dilution buffer10 mM MgCl2/10 mM CaCl2

NZC medium (UNIT 1.1)Beckman JA-20 rotor or equivalent

1. Grow an overnight culture of a lambda-sensitive strain of E. coli in LB medium at 37°C.

Lambda-sensitive strains will support lytic growth. This can be tested by spotting 10 �l ofa lambda lysate onto a lawn of bacteria. If the strain is lambda-sensitive, a plaque willform where the phage were spotted.

2. Using a sterile toothpick or capillary tube, pick a single plaque (see step 3 of basicprotocol), blow it into a tube that contains 0.4 ml lambda dilution buffer, and placetube at 4°C for 2 hr to allow phage to elute.

Alternatively, 105 to 108 phage from a liquid lysate or plate stock can be used.

3. Combine 0.1 ml eluted phage with 0.1 ml of saturated culture and 0.1 ml of 10 mMMgCl2/10 mM CaCl2 solution and incubate 15 min in a 37°C water bath.

Incubating with Mg++ and Ca++ allows the phage to adsorb to the bacteria.

4. Transfer this solution to 50 ml of NZC medium and shake vigorously at 37°C untillysis occurs (usually between 6 and 8 hr).

Good aeration is important for high yields.

5. The culture should be checked frequently after 6 hr, and harvested immediately uponclearing.

6. Add a few drops of chloroform to lyse any remaining cells, transfer the solution toCo-rex or Nalgene tubes (being careful to leave the chloroform behind), and spin 10min at 10,000 rpm (12,100 × g), 4°C, to pellet the cell debris.

7. Save as much of the lysate as desired. Transfer to a screw-cap tube, add a few dropsof chloroform, vortex briefly, and store at 4°C.

The titer of the phage should be determined as described in UNIT 1.11.

SUPPORTPROTOCOL

STORING PHAGE LYSATES

Phage stocks should be stored in SM (UNIT 1.11) plus a few drops of chloroform at 4°C.Screw-cap glass tubes with rubber-lined or teflon caps, capable of holding at least 1 ml, aregenerally used, although disposable plastic tubes work well. Lambda titers drop over a periodof several years. Stocks should be dated and checked occasionally to determine phage titer.Addition of 0.1% gelatin to the SM slows down the rate at which phage titers drop whenstored under these conditions. Lysates can also be stored frozen in 15% glycerol.


Lambda dilution buffer20 mM Tris⋅Cl, pH 820 mM MgCl2


1.12.2


COMMENTARY

Background InformationPlate lysis is typically used to make small

phage stocks, which are then used as interme-diates for making larger stocks for preparationof phage DNA. However, if agar is replaced byagarose in the plate recipe and in the top agar(see UNIT 1.1), then DNA extracted from phagein the lysate is an acceptable substrate for re-striction enzymes and ligases.

Liquid lysates generally give slightly loweryields than plate lysates. Less work is required,however, especially when large quantities ofphage are needed. This fact, taken together withthe fact that there is no contaminating agar inthe lysate, makes this method preferable forisolating phage for DNA preps. In the liquidlysis procedure, cells are diluted into NZCmedium after phage adsorption. This is a richmedium that contains amino acids but no glu-cose or other sugars. In cells grown in thismedium, sugar receptors like LamB protein arefractionally induced (so phage can adsorb to thecell), but cells are not covered with so manyreceptors that adsorption to their debris de-pletes the yield of phage from the lysate. Inaddition, cells grown on this medium seem tocause infecting phage to grow lytically, whichhelps to obtain high titers of turbid phages.

Critical ParametersLB medium and top agar can replace lambda

plates and top agar for the plate lysis method,but the phage yield is usually lower. It is impor-tant that the plates be freshly poured, and thatthe top agar be freshly melted. The plates donot even have to be dry; a loose lawn is noproblem as long as the plates are not tilted. Ifthe yield is less than expected, it may be becausethe plates were not fresh, or because the par-ticular phage used does not give a burst as largeas that given by wild-type lambda. The size ofsingle plaques made by the phage sometimesgives a clue that the second problem obtains. Ifplaques made by the phage in question aresmaller than those made by closely relatedphages, it is often worthwhile to reduce thenumber of starting cells, or to increase thestarting number of phage by combining severalplaques, and to incubate the plates longer until

the lawn is completely lysed.In the liquid lysis method, good aeration is

essential for high yields. Flasks should be nomore than 1⁄5 full. Cultures should be watchedcarefully as they approach lysis and harvestedsoon after lysis is complete to prevent releasedphage from reinfecting cell debris. Lysis usu-ally takes between 6 and 8 hr. If there is no signof lysis after 8 hr, there probably will not beany, and one can either go on to add the chlo-roform and complete the procedure, or abandonhope and start over.

Anticipated ResultsThe plate lysis procedure yields ∼10 ml of

a phage stock containing 1010 to 1011 phage/ml. The liquid lysis procedure usually yields astock with 5 × 109 to 3 × 1010 phage/ml.

Time ConsiderationsThe plate lysate procedure usually takes about

8 hr. The liquid lysate procedure takes 2 hr to elutethe phage from the plaque, and 6 to 9 hr betweenthe time the cells are infected and the time thelysate is harvested. The time required to elute thephage is very flexible. Two hours will ensure thatmore than 90% of the phages are eluted from theplaque; shorter elution times allow less phage tobe eluted. Elution times as short as 30 min gen-erally work but do not always give good lysis.Phage may be eluted for longer periods of time;overnight is often convenient. Once the host cellsare infected, it takes ∼6 to 9 hr for lysis andpreparation of the lysate.

Key Reference


Origin of this liquid lysate procedure.



1.12.3


UNIT 1.13Preparing Lambda DNA from Phage Lysates

DNA extracted from lambda-derived vectors is typically subcloned into plasmid orfilamentous phage vectors. The first two protocols describe methods for isolating phageDNA from large- and medium-scale liquid lysates. These two methods use eitherdensity-gradient centrifugation or ion-exchange chromatography to purify the phageparticles. The third protocol describes a rapid procedure for isolating phage DNA, suitablefor small-scale liquid lysates.

BASICPROTOCOL

PREPARING DNA BY STEP- AND EQUILIBRIUM-GRADIENTCENTRIFUGATION

A scaled-up liquid lysate is used to prepare a large quantity of highly purified phage DNA.Phage is separated from cellular debris by a CsCl step gradient followed by equilibrium-gradient centrifugation. Two alternate sets of steps are provided for extracting λ DNAfrom CsCl-purified phage particles. In the first method, CsCl is removed by dialysis andthe DNA is extracted by phenol and chloroform. In the second approach, phage DNA isextracted directly from CsCl-purified phage particles with high-grade formamide.

Materials

5× polyethylene glycol (PEG) solutionSuspension medium (SM; UNIT 1.11)Potassium chlorideCsCl solutionsLow-salt bufferBuffered phenol (UNIT 2.1)Chloroform2 M Tris⋅Cl (pH 8.5)/0.2 M EDTA (optional, for formamide extraction)Formamide (very high grade, preferably recrystallized; optional)TE buffer, pH 8.0 (APPENDIX 2)

Beckman JA-10, JA-20, SW-28, and VTi50 rotors and bottles/tubes (or equivalents)3-ml syringe with 25-G needleBeckman VTi50 quick-seal tubes

Additional materials for preparing liquid phage lysate (UNIT 1.12), titering lambdaphage (UNIT 1.11), and quantitation of DNA (APPENDIX 3)

Prepare and concentrate the phage1. Use 25 ml liquid lysate to make a 1000-ml lysate (see UNIT 1.12).

2. Split the lysate into two JA-10 centrifuge bottles and spin 10 min at 10,000 rpm(17,700 × g), 4°C, to remove cell debris.

3. Transfer supernatant to a 1000-ml graduated cylinder and add 5× PEG solution to afinal concentration of 1×. Invert gently to mix. Let sit overnight at 4°C.

The PEG solution causes the phage to precipitate.

4. Remove ∼50 ml of supernatant and save for step 5. Pour off remaining supernatant,being careful not to lose any of the white precipitate.

5. Transfer precipitate to Nalgene centrifuge tube. Rinse cylinder with saved super-natant and transfer to centrifuge tube. Spin 10 min in JA-20 rotor at 5000 rpm (3000× g), 4°C.

Supplement 10

Contributed by Karen LechCurrent Protocols in Molecular Biology (1990) 1.13.1-1.13.10Copyright © 2000 by John Wiley & Sons, Inc.

1.13.1


6. Place centrifuge tubes on ice. Remove the top layer, being careful not to remove anyof the thick white phase, which contains the PEG solution and the phage.

7. Resuspend the white phase in a minimum volume of SM. Transfer to a 125-ml flask.

The volume of the suspension medium added should not be more than three times the volumeof the white phase.

8. Determine the amount of solid KCl needed to make a 1 M solution. Add this amountof KCl in four aliquots of approximately equal size, mixing well after each addition.Let sit on ice for 15 to 30 min.

Adding KCl precipitates the PEG solution slowly while leaving the phage behind.

9. Transfer to Nalgene centrifuge tube and spin 10 min in a JA-20 rotor at 10,000 rpm(12,100 × g), 4°C.

The PEG solution will be pelleted and the phage will remain in the supernatant.

10. Measure the phage titer and keep the supernatant in a 16- or 18-mm glass test tube.

The phage titer should be ∼1 × 1012 to 5 × 1013 pfu/ml.

Isolate the phage particles11. Pour a CsCl step gradient in an SW-28 centrifuge tube as follows:

First layer: 3.5 ml CsCl solution, d = 1.7 g/mlSecond layer: 2.5 ml CsCl solution, d = 1.5 g/mlThird layer: 2.5 ml CsCl solution, d = 1.3 g/ml

The layers must be added very slowly to avoid mixing.

12. Carefully layer the lambda lysate (supernatant from step 10) on top of this gradient(see sketch 1.13A).

13. Fill tube to just below the top with SM. Spin 2 hr in an SW-28 rotor at 24,000 rpm(104,000 × g), 4°C.

14. Recover the phage band by inserting a 3-ml syringe with a 25-G needle into the sideof the tube just below the band and drawing it into the syringe (see Fig. 1.7.1 inCsCl/EtBr plasmid prep, UNIT 1.7).

Usually, three bands are visible: one blue phage band at the lowest gradient, one blue


resuspension mediumor mineral oil

phage lysate

d = 1.3 g /ml

d = 1.5 g /ml

d = 1.7 g /ml

CsCI step gradient

Sketch 1.13A

1.13.2

PreparingLambda DNA from

Phage Lysates

band containing empty phage heads, and one white cell debris band. Occasionally one ortwo of these bands are missing. This is not a problem as long as the band that is visible isin the d = 1.5 g/ml layer. If no band is visible, there are probably not enough phage to makeit worthwhile continuing the procedure. If the layers are not clearly defined, but a bluishband appears at about the right place, recover the band and determine its density byweighing 100 �l of the lysate. If its density is ∼1.5, it probably is phage.

15. Transfer the phage to Beckman VTi50 quick-seal tubes. Fill tubes with CsCl solution(d = 1.5 g/ml).

About half the weight of the phage particle is protein; the other half is DNA. Phage DNAis denser than phage proteins. Thus, phages with genomes larger than wild-type are denserthan phages with wild-type-length genomes, and phages with smaller genomes are lessdense.

16. Spin 24 hr in VTi50 rotor at 30,000 rpm (81,500 × g), 4°C.

17. Remove the band as shown in Figure 1.7.1 of CsCl/EtBr plasmid prep (UNIT 1.7), usinga 3-ml syringe and a 25-G needle. Only one band should be visible.

Extract the phage DNATwo methods are presented below for extracting DNA from purified phage. In steps 18ato 21a, cesium chloride is removed by dialysis prior to extraction of DNA with phenoland chloroform. In steps 18b to 21b, DNA is extracted directly from the isolated phagewith high-grade formamide. The latter approach is quicker and possibly gentler.

Dialysis and phenol/chloroform extraction:

18a. Dialyze, with stirring, in 500 ml low-salt buffer for ≥4 hr at 4°C. Repeat twice.

19a. Extract three times by agitating gently for 20 min with an equal volume of bufferedphenol.

20a. Extract twice using an equal volume of chloroform.

21a. Dialyze, with stirring, in 500 ml TE buffer, pH 8.0, for 8 hr at 4°C. Change bufferonce. Proceed to step 22.

Residual phenol and chloroform are removed by dialysis rather than ethanol precipitationbecause purified phage DNA is very difficult to resuspend.

Formamide extraction:

18b. Measure the volume of phage band from step 17. Add 0.1 vol of 2 M Tris⋅Cl (pH8.5)/0.2 M EDTA and invert to mix.

19b. Add 1 vol formamide, mix, and let stand 30 min at room temperature.

20b. Add 2 vol (each equal to the original volume of phage band in step 18b) of 100%ethanol at room temperature. Mix gently and microcentrifuge 1 to 2 min.

21b. Discard supernatant and rinse pellet with 70% ethanol. Remove all droplets ofethanol with a drawn-out pipet and dissolve the moist pellet in TE buffer, pH 8.0.

22. Measure DNA concentration as described in APPENDIX 3.


1.13.3


ALTERNATEPROTOCOL

PREPARING DNA USING DEAE-CELLULOSECOLUMN CHROMATOGRAPHY

This protocol employs an ion-exchange resin to preferentially bind contaminants in crudephage lysate (E. coli DNA, RNA, and protein) while phage particles pass through thecolumn. The result is a highly purified phage preparation. The DNA is then extracted fromthe purified phage with organic solvents and precipitated with ethanol. The method issimple, rapid, and does not require DNase, RNase, or CsCl density gradient centrifugationof either phage particles or DNA. The high-quality λ DNA obtained is suitable for cloning,sequencing, restriction enzyme digestion, ligation, and in vitro packaging.

Materials

TM buffer2% sodium dodecyl sulfate (SDS; optional)0.1 M EDTA (optional)Sodium chloridePolyethylene glycol (PEG) 8000DEAE-cellulose (microgranular anion exchanger; Whatman DE52 #4057-050)0.05 N HCl10 M NaOHSodium azide5 M NaClIce-cold 100% isopropanolTE buffer, pH 8.0 (APPENDIX 2)

25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1)

3 M sodium acetate, pH 6.070% ethanol and ice-cold 100% ethanol

Beckman JA-14 and JA-20 rotors (or equivalents)15- and 30-ml Corex centrifuge tubes10-ml disposable syringe (1.4 cm-i.d., optional; Becton Dickinson) or

1.5 × 10–cm standard glass or disposable column (Bio-Rad)Glass-fiber filter or glass wool (optional)

Additional reagents and equipment for preparing liquid or plate lysate (UNIT 1.12),titering lambda phage (UNIT 1.11), agarose gel electrophoresis (UNIT 2.5), andphenol extraction/ethanol precipitation (UNIT 2.1)

Prepare concentrated crude phage lysate1. Prepare liquid or plate lysate and determine the phage titer. The lysate should contain

1-2 × 1010 pfu/ml in a volume of 200 ml.

When using plate lysates, collect phage in TM buffer (instead of SM) from six big (150 mm)or fifteen small (90 mm) petri dishes and adjust volume to 200 ml.

Alternatively, a 0.7% agarose minigel can be used to detect λ DNA in phage lysates. Treat20 �l lysate with 2 �l of 2% SDS and 2 �l of 0.1 M EDTA for 5 min at room temperature.Load onto gel. Include size standard and control (nontreated) lanes. In this gel, theSDS/EDTA-treated lysate sample should show a distinct λ DNA band compared to thenontreated sample (Fig. 1.13.1).

2. Add 5.8 g NaCl (0.5 M final) and 20 g PEG 8000 (10% wt/vol final) to 200 ml phagelysate.

If the phage titer is low, it may be necessary to add NaCl first to release phage adheringto the debris. Remove debris by centrifuging 10 min in JA-14 at 6000 rpm (5500 × g), 4°C,and then add PEG. Dissolve the PEG flakes by gentle stirring and place on wet ice 1 hr.This is enough time to precipitate most of the phage.


1.13.4


Phage Lysates

3. Separate precipitated phage by centrifuging 10 min in a JA-14 rotor at 6000 rpm,4°C.

Drain liquid from the bottles by placing in an inverted position; and shake by hand toremove any remaining liquid.

4. Resuspend phage pellet in 3 ml TM buffer and transfer to a 15-ml Corex centrifugetube. Add 3 ml chloroform, mix gently, and centrifuge 10 min in a JA-20 rotor at 5000rpm (3000 × g), 4°C. Collect the phage-containing upper aqueous phase withoutdisturbing the PEG interface.

Corex tubes make it easy to see the PEG interface and the well-separated supernatant.

5. Add 3 ml TM buffer to the tube, mix, and centrifuge as in step 4. Save the aqueousfraction and combine with aqueous fraction from step 4. Adjust the volume to 6 mlwith TM buffer.

This step removes phage particles trapped at the PEG interface.

Prepare the DEAE-cellulose column6. Prepare a slurry of DEAE-cellulose by adding several volumes of 0.05 N HCl. Make

sure the pH of the solution is below 4.5.

It is convenient to prepare enough DEAE-cellulose for 30 to 40 columns. Each columnrequires ∼9 ml resin (for 200 ml phage lysate).

7. While stirring, add 10 M NaOH until the pH approaches 7.5.

8. Let the resin settle, decant or aspirate, and equilibrate the resin with TM buffer (seemanufacturer’s instructions).

Make sure the DEAE-cellulose is completely equilibrated with TM buffer. This may requirerepeating step 8 four or five times. This process also removes fine particles from the resin.

9. Adjust the slurry to 75% resin and 25% TM buffer.

For long-term storage (several months), add NaN3 to a final concentration of 0.02% andplace at 4°C.

10. Pour 9 ml resin in a 10-ml disposable syringe (1.4-cm i.d.) or a 1.5 × 10–cm column.This will result in a bed height of 5 to 6 cm, appropriate for 200 ml of phage lysate.


1 2 3

λDNA

Figure 1.13.1 Agarose minigel of bacteriophage lambda lysates.Lane 1: λ DNA digested with HindIII.Lane 2: 20 µl of lysate without SDS and EDTA.Lane 3: 20 µl of lysate treated with 2% SDS and 0.1 M EDTA.

1.13.5


When using a disposable syringe, place a glass-fiber filter or glass wool at the bottom ofthe syringe to support the resin. Attach a small piece of tubing with a stopcock to the syringetip to control the flow. Column preparation is easier with a standard glass or disposablecolumn. See UNIT 10.10 for complete discussion of ion-exchange chromatography and Figure3.4.1 for a sketch of homemade column setup.

Isolate the DNA11. Load 6 ml crude phage solution from step 5 onto DEAE-cellulose column and elute

with 10 ml TM buffer. Discard the first 3 ml of the eluate (void volume); collect thenext 13 ml in a 30-ml Corex tube.

The 13-ml eluate may be collected in 1-ml fractions, if desired. Generally, phage eluteimmediately after the void volume. Fractions containing high-titer phage appear bluishdue to light scattering. This indicates that the column is working well. Phage purificationcan also be confirmed by analyzing 5 �l of each fraction on an agarose minigel (Fig. 1.13.1).

12. Add 2 ml of 5 M NaCl (400 mM final) and 10 ml of ice-cold 100% isopropanol (40%final) to the 13-ml eluate. Place at −20°C for 15 min.

13. Centrifuge 10 min in JA-14 rotor at 5000 rpm (3800 × g), 4°C, and discardsupernatant.

Invert centrifuge tubes until liquid is completely drained. Using tissue paper, wipe the wallsof the centrifuge tube without touching the phage pellet. The pellet will appear as a featherydesign adhering to wall of the tube. If there is not a sufficient pellet, abandon the experimentand start again from new lysate.

14. Resuspend the phage pellet in 0.8 ml TE buffer, pH 8.0, and divide equally into two1.5-ml microcentrifuge tubes.

15. Extract once with phenol, twice with phenol/chloroform/isoamylalcohol, and finallywith chloroform. Repeat extractions until no visible protein precipitate is seen at theinterface.

16. Precipitate λ DNA by adding 1⁄10 vol of 3 M sodium acetate, pH 6.0, and 2 vol ice-cold100% ethanol.

In general, λ DNA forms a fibrous precipitate immediately after the addition of ice-coldethanol at room temperature. A white cloudy precipitate indicates leftover protein. The λDNA may be spooled out with a glass rod or condensed into a globule by gentle mixing.

17. Rinse the DNA pellet with 70% ethanol, air dry, and resuspend in TE buffer, pH 8.0.After dissolving completely, measure DNA concentration (UNIT 1.7) and adjust to 200µg/ml in TE buffer. Store at 4°C.

It may take several hours for the λ DNA to go into solution.


1.13.6


Phage Lysates

ALTERNATEPROTOCOL

PREPARING DNA FROM SMALL-SCALE LIQUID LYSATES

This protocol is useful for making small quantities of DNA to be used for restrictionanalysis. Phage are concentrated by centrifugation and their capsids are destroyed withphenol. The DNA is then ethanol precipitated.


5 mg/ml DNase (UNIT 3.12)10 mg/ml DNase-free RNase (UNIT 3.13)0.05 M Tris⋅Cl, pH 8.03 M sodium acetate, to pH 4.8 with acetic acid

1. To approximately 50 ml liquid phage lysate (UNIT 1.12), add 10 µl of 5 mg/ml DNaseand 25 µl of 10 mg/ml DNase-free RNase. Incubate 1 hr at 37°C.

This treatment will degrade the bacterial DNA and RNA released during lysis. The viscosityof the mixture should decrease.

2. Centrifuge 11⁄2 hr at 27,000 rpm in an SW-28 rotor (132,000 × g), 4°C.

Alternatively, pellet the phage by spinning 21⁄4 hr in JA-20 rotor at 20,000 rpm (48,000 ×g), 4°C. The pellet obtained in this manner will resuspend somewhat more easily.

3. Discard supernatant. Invert the tubes on an absorbent surface, e.g., paper towels, toremove any remaining liquid. Resuspend the phage pellet in 200 µl of 0.05 M Tris⋅Cl,pH 8.0.

A small translucent pellet should be visible after the tubes are inverted.

4. Transfer the solution to a microcentrifuge tube and add 200 µl buffered phenol. Vortex20 min or shake 20 min in microcentrifuge tube shaker. Spin 2 min in microcentrifugeand save the aqueous (top) layer. Repeat phenol extraction.

Phenol denatures the phage capsids and releases the DNA. This denatured capsid proteinappears as a thick white precipitate at the phenol/water interface. Vigorous agitation isnecessary because the pellet is difficult to resuspend.

There should be less white precipitate after the second phenol extraction. If there is still alarge amount at the interface, do a third extraction.

5. Add 200 µl chloroform, shake well, and spin in microcentrifuge briefly. Save theaqueous (top) layer. Repeat.

6. Add 20 µl of 3 M sodium acetate, pH 4.8, and precipitate DNA with 2 vol of 100%ethanol at room temperature. Spin in microcentrifuge for 10 min.

7. Remove supernatant. Wash pellet by adding 1 ml of 70% ethanol and spinning 5 min.

8. Remove supernatant. Dry pellet under a vacuum and resuspend the DNA in 100 µlTE buffer, pH 8.0.

The DNA will resuspend more quickly if the pellet is still slightly wet.

3 �l of the DNA suspension should be used for a restriction digest.


1.13.7



CsCl solutionsd = 1.3 g/ml: 31.24 g CsCl + 68.76 ml H2Od = 1.5 g/ml: 45.41 g CsCl + 54.59 ml H2Od = 1.7 g/ml: 56.24 g CsCl + 43.76 ml H2O

The equation for preparing the CsCl solutions is: % w/w = 137.48 − 138.11/d. Thiscalculation assumes the CsCl has no water in it. Typically, CsCl from the shelf will haveadsorbed water from the air, and the densities of these stock solutions will be lower thanclaimed. However, in our experience, the phage band is always found in the middle layer ofthe step gradient.

Low-salt buffer0.05 M NaCl0.05 M Tris⋅Cl, pH 7.50.01 M MgSO4

5× polyethylene glycol (PEG) solution, 600 ml207 g Carbowax (PEG 6000)6 g dextran sulfate49.5 g NaCl350 ml H2O

TM buffer50 mM Tris⋅Cl, pH 7.510 mM MgSO4

COMMENTARY

Background InformationThis unit provides three methods for extract-

ing and purifying DNA from intact phage par-ticles. The three methods differ in the amountof starting material or phage lysate, and in themanner in which phage are concentrated andpurified before lysing and DNA release.

The large-scale preparation (basic protocol) isuseful when large quantities of highly purifiedDNA (over 200 µg) are required—for example,in the construction of libraries. It is based ontraditional methods of bacteriophage lambda iso-lation, involving precipitation of phage from thelysate by polyethylene glycol (PEG) and sub-sequent purification by CsCl step and equilibriumdensity-gradient centrifugation (Yamamoto et al.,1970; Davis et al., 1980; Maniatis et al., 1982). Itis necessary to use two different gradients topurify phage away from the considerable amountof cellular debris present in a large lysate. Follow-ing phage purification, DNA is extracted fromcapsids by one of two approaches. In one method,phage proteins are removed by a series of phenolextractions and resulting phage DNA is dialyzedin TE buffer to remove any remaining phenol orchloroform. In the other method, DNA is ex-tracted directly from the capsids with high-gradeformamide.

The first alternate protocol describes a mod-erate-scale procedure, capable of yieldingenough pure lambda DNA (about 200 µg) formost standard DNA manipulations, such ascloning, sequencing, in vitro packaging, andSouthern blotting. Phage particles are separatedfrom cellular components in the lysate by ion-exchange chromatography, resulting in a highdegree of purification in a single step. Thechromatographic purification works as follows.DEAE (diethyl aminoethyl)-cellulose is an an-ion-exchange resin—i.e., it has positivelycharged groups that adsorb negatively chargedmolecules in buffer of near neutral pH andmedium ionic strength. The major cellular con-taminants in bacteriophage lysates are nega-tively charged (polyanionic) molecules (DNA,RNA, and proteins) which are preferentiallyadsorbed onto the positively charged groups ofDEAE-cellulose (Creaser and Taussig, 1957;Benson and Taylor, 1984; Shuang-Young,1986).

Two different ways to use column chroma-tography with Whatman DE52 anion exchangeresin for phage purification have been reported.In the procedure described here, cellular con-taminants are bound to the column, leav-ing phage particles free (Reddy et al., 1988;


1.13.8


Phage Lysates

White and Rosenzweig, 1989). Because SDSand proteinase K are not used in the nucleic acidextraction, the resulting DNA can be used withrestriction endonucleases and DNA-modifyingenzymes without further purification, and hasbeen used successfully to make genomic ex-pression libraries (Webb et al., 1989). Alterna-tively, binding phage particles to the resin withsubsequent elution using a high-salt buffer(Helms et al., 1985) is used for processingseveral clones simultaneously to obtain a fewmicrograms of DNA for subcloning.

The second alternate protocol describes arapid, small-scale method for phage purifica-tion which yields small amounts of moderatelypure DNA, suitable for restriction analysis. Inthis procedure, phage are pelleted out of solu-tion with a high-speed spin, and host nucleicacids are destroyed using RNase and DNase.(These enzymes have no effect on phage DNAas it is still packaged in phage heads.) PhageDNA is subsequently extracted from the cap-sids with phenol and is concentrated with etha-nol precipitation.

Critical ParametersRegardless of procedure, most failures to

isolate lambda DNA are due to low phage titerin the initial lysate. Thus, hours of wasted timecan be spared by first titering the phage lysate.Phage can be titered by dilution plating (UNIT

1.11); if the titer is less than 109 pfu/ml, a newlysate should be made. An alternative and usu-ally superior method to determine the amountof DNA is to treat 20 µl of lysate withSDS/EDTA followed by electrophoresis on anagarose minigel (UNIT 2.5). The gel assay takesless time and provides a more accurate estima-tion of DNA present in both viable and nonvi-able phage particles. Figure 1.13.1 (lane 3)shows a typical gel resolving relative amountsof cellular nucleic acids and λ DNA (∼50 ng)present in a lysate. One should be able to visu-alize ≥10 ng of λ DNA in the band. If there isno visible band, it is advisable to start fromfresh lysate.

In chromatographic purification, the ratio oflysate to column size is an important variable.A 9-ml bed volume is sufficient for 200 ml ofliquid lysate (at 1010 pfu/ml); if more lysate isused (or, if lysate has more highly concentratedcellular debris), the bed volume should be in-creased proportionately. If the column is over-loaded, cellular DNA and RNA may coelutewith the phage (to assess this, monitor columnfractions with sample of crude lysate by run-ning on an agarose gel). If DNA is contami-

nated, column fractions can be pooled andtreated with DNase and RNase (UNITS 3.12 & 3.13)before organic solvent extraction of phageDNA. One should carefully prepare, equili-brate, and pack DEAE-cellulose according toinstructions in the Whatman information leaflet(see also UNIT 10.10). Note that the type of DEAE-cellulose (Whatman DE52), as well as the pHand ionic strength of the buffer (use TM bufferrecipe provided), are important. Chloroform-lysed cultures often contain very viscous, high-molecular-weight chromosomal DNA whichcan block the DEAE column. Such a lysate canbe treated with DNase before loading onto thecolumn.

After organic extraction, it may be prefer-able to dialyze rather than precipitate phageDNA, because large DNA can be difficult toresuspend. Lambda DNA is large and easilysheared after it is isolated. When intact DNA isdesired (e.g., when phage arms are to be pre-pared for making a library), the DNA shouldbe treated with special care: shaken, swirled, orvortexed very gently to mix.

Anticipated ResultsYield of λ DNA from each of these proce-

dures depends upon the phage titer and theoriginal lysate volume. If the initial lysate titeris around 1-2 × 1010 pfu/ml, then 1 ml of phagelysate ought to yield ∼1 µg of DNA; that is, 2× 1010 phage particles gives ∼1 µg purifiedphage DNA. Thus, the large-scale procedure,starting with 1000 ml of lysate, gives ∼1 mg ofphage DNA; the DEAE-column procedure(200 ml lysate) results in ∼200 µg DNA; andthe small-scale procedure (50 ml lysate), pro-vides ∼50 µg DNA.

Time ConsiderationsThe large-scale prep employing gradient pu-

rification typically takes at least 3 days: ∼30min the first day to set up the PEG precipitation;∼5 hr the next day to spin down and collect theprecipitate, run the step gradient, and load theequilibrium gradient; and ∼30 min the third dayto collect and phenol extract the phage band,followed by two dialysis steps lasting 8 hr. Thephage lysates made as intermediates can bestored for months at 4°C without decreasing intiter. The small-scale prep takes ∼4 to 6 hr andit is fairly easy to process a number of samplesat once.

Column-purified phage and DNA isolationcan be completed in 4 hr, starting from liquidor plate lysate. Preparation of the crude phagesolution for the column takes ∼2 hr. Another 2


1.13.9


hr are needed for column setup, column load-ing, elution, and DNA extraction. It is usuallymost convenient to process four to five differentcolumns simultaneously. The DEAE-celluloseslurry can be prepared in advance and stored at4°C for several months.

Literature CitedBenson, S. and Taylor, R.K. 1984. A rapid small-

scale procedure for isolation of phage lambdaDNA. Biotechniques 2:126-127.

Creaser, E.H. and Taussig, A. 1957. The purificationand chromatography of bacteriophages on an-ion-exchange cellulose. Virology 4:200-208.

Helms, C., Graham, M.Y., Dutchik, J.E., and Olson,M.V. 1985. A new method for purifying lambdaDNA from phage lysates. DNA 4:39-49.

Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982.Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Laboratory, Cold Spring Harbor,N.Y.

Reddy, K.J., Kuwabara, T., and Sherman, L.A. 1988.A simple and efficient procedure for the isolationof high-quality phage λ DNA using a DEAE-cel-lulose column. Anal. Biochem. 168:324-331.

Shuang-Young X. 1986. A rapid method for prepar-ing phage λ DNA from agar plate lysates. GeneAnal. Techn. 3:90-91.

Webb, R., Reddy, K.J., and Sherman, L.A. 1989.Lambda ZAP: Improved strategies for expres-sion library construction and use. DNA 8:69-73.

White, B.A. and Rosenzweig, S. 1989. A reliablemethod for the purification of bacteriophage λDNA. Biotechniques 7:694-695.

Key References

PEG precipitationYamamoto, K.R., Alberts, B.M., Benzinger, R.,

Lawhorne, L., and Treiber, G. 1970. Rapid bac-teriophage sedimentation in the presence ofpolyethylene glycol and its application to large-scale virus purification. Virology 40:734-744.

Step gradient and equilibrium gradientDavis, R.W., Botstein, D., and Roth, J.R. 1980. A

Manual for Genetic Engineering: AdvancedBacterial Genetics, pp. 70-113. Cold Spring Har-bor Laboratory, Cold Spring Harbor, N.Y.

Includes a number of other protocols for prepara-tion of DNA from lambda.

Ion-exchange purificationReddy et al. 1988. See above.

Describes the details of the DEAE-cellulose columnchromatography of λ DNA upon which this protocolis based. Also describes a large-scale isolation pro-cedure of λ DNA from a 1-liter liquid lysate using a45-ml bed volume DEAE column.


K.J. Reddy and L.A. Sherman (column purification)Purdue UniversityWest Lafayette, Indiana


1.13.10


Phage Lysates

SECTION IVVECTORS DERIVED FROMFILAMENTOUS PHAGES

UNIT 1.14Introduction to Vectors Derived fromFilamentous Phages

Many vectors in current use are derived fromfilamentous phages. These vectors are usedbecause DNA inserted into them can be recov-ered in two forms—double-stranded circlesand single-stranded circles. Foreign DNA isinserted into double-stranded vector DNA and,then, reintroduced into cells by transformation.Once inside the cells, double-stranded DNAreplicates, giving rise both to new double-stranded circles and to single-stranded circlesderived from one of the two strands of thevector. Single-stranded circles are packagedinto phage particles and secreted from cells(which do not lyse). Centrifugation of a cultureof infected cells yields a supernatant that is fullof particles containing only a single strand ofthe phage DNA. This ready availability of sin-gle-stranded DNA has made possible new pro-

cedures for sequencing DNA (Chapter 7), mu-tagenesis (Chapter 8), and other techniquesdescribed in this book.

Techniques to isolate double-stranded DNAusing these vectors are described in UNIT 1.15.

DEVELOPMENT AND USE OFFILAMENTOUS PHAGE VECTORS

Prototypes of the filamentous phage vec-tors are the M13mp derivatives (see Figs.1.14.1 and 1.14.2). These vectors were devel-oped by Joachim Messing and his co-work-ers, who also developed and disseminated sim-ple and powerful techniques for working withthem. The M13mp vectors are viable phages.Foreign DNA is inserted into a polylinker (astretch of DNA that contains contiguous re-striction sites) located in an inessential region

SalIAccIHincII

(Met)ThrMetIleThrAsp lac Z′1

EcoRl Sst l Xma lSma l

Bam HI XbaI PstI SphI HindIIIKpn l

6230 6250 6270 6290

ATG ACC ATG ATT ACG AAT TCG AGC TCG GTA CCC GGG GAT CCT CTA GAG TCG ACC TGC AGG CAT GCA AGC TTG GCA CT

6

2 10

4

1

3

8

7

9

5

polylinker

+ strand

M13mp187250bp

lacZ′

′laclSnaBI 1268

Bsml 1746

Nar l 6001Ahall 6001

Avall 5914

Dralll 5716

Nael 5613Cfr10l 5613

Bal l 5080

ApaLl 4743

Pvul 6405 Fspl 6425

Bgl l 6431Mst ll 6508 0

Bgl ll 6935

EcoRl Sac l Kpn l Smal BamHI XbaI

Sal IHincIIAccI Pst I SphI HindIII

62826231

ori– strand

Figure 1.14.1 M13mp18. M13mp18 is one of the M13mp vectors made by Messing and col-leagues. Insertion of DNA into the polylinker inactivates the lacZ alpha fragment. When insert-con-taining phages are plated under appropriate conditions (UNIT 1.15), they form colorless plaques;vectors that do not contain inserts form blue plaques (Yanisch-Perron et al., 1985, and referencestherein).

Supplement 13

Contributed by David Greenstein and Roger BrentCurrent Protocols in Molecular Biology (1990) 1.14.1-1.14.5Copyright © 2000 by John Wiley & Sons, Inc.

1.14.1



Sal lAccI

HincII

M13mp8�pUC81

THRACC

2METATG

3ILEATT

4THRACG

5ASNAAT

6SERTCC

1arg

CGG

2gly

GGA

3ser

TCC

4val

GTC

5aspGAC

6leu

CTG

7gln

CAG

8pro

CCAATG

9ser

AGC

10leu

TTG

11ala

GCA

7LEUCTG

8ALAGCC

HindIIlPst lBamHIXmalSmal

EcoRI HaellI

Sal lAccI

HincII

Sal lAccI

HincII

Sal lAccI

HincII

M13mp19�pUC191

THRACC

2METATG

3ILEATT

4THRACG

1pro

CCA

2ser

AGC

3leu

TTG

4his

CAT

5ala

GCC

6cys

TGC

7arg

AGG

8ser

TCG

9thr

ACT

10leu

CTAATG

11glu

GAG

12aspGAT

13pro

CCC

14arg

CGG

15val

GTA

16pro

CCG

17ser

AGC

18ser

TCG

5ASNAAT

6SERTCA

7LEUCTG

8ALAGCC

HindllI SphI Pst l Sal lHincIIAccI

Xbal BamHI XmalSmal

Kpn l Sst l EcoRI HaellI

M13mp18 �pUC181

THRACC

2METATG

3ILEATT

4THRACG

5ASNAAT

6SERTCG

1ser

AGC

2ser

TCG

3val

GTA

4pro

CCC

5gly

GGG

6aspGAT

7pro

CCT

8leu

CTAATG

9glu

GAG

10ser

TCG

11thr

ACC

12cys

TGC

13arg

AGG

14his

CAT

15ala

GCA

16ser

AGC

17leu

TTG

18ala

GCA

7LEUCTG

8ALAGCC

HindIIlSphIPst lXbalBamHIXmalSmal

KpnlSst lEcoRI HaellI

M13mp11�pUC131

THRACC

2METATG

3ILEATT

4THRACG

1pro

CCA

2ser

AGC

3leu

TTG

4gly

GGC

5cys

TGC

6arg

AGG

7ser

TCG

8thr

ACT

9leu

CTA

10glu

GAGATG

11aspGAT

12pro

CCC

13arg

CGG

14ala

GCG

15ser

AGC

16ser

TCG

5ASNAAT

6SERTCA

7LEUCTG

8ALAGCC

HindIlI Pst l Xbal BamHI XmalSmal

Sst l EcoRI HaellI

M13mp10�pUC121

THRACC

2METATG

3ILEATT

4THRACG

5ASNAAT

6SERTCG

1ser

AGC

2ser

TCG

3pro

CCC

5aspGAT

6pro

CCT

7leu

CTA

8glu

GAGATG

9ser

TCG

10thr

ACC

11cys

TGC

12ser

AGC

13pro

CCA

14ser

AGC

15leu

TTG

16ala

GCA

7LEUCTG

8ALAGCC

HindIIlPst lSal lAccI

HincII

XbalBamHIXmalSmal

Sst lEcoRI HaellI

4gly

GGG

M13mp9�pUC91

THRACC

2METATG

3ILEATT

4THRACG

1pro

CCA

2ser

AGC

3leu

TTG

4ala

GCT

5ala

GCA

6gly

GGT

7arg

CGA

8arg

CGG

9ile

ATC

10pro

CCCATG

11gly

GGG

5ASNAAT

6SERTCA

7LEUCTG

8ALAGCC

HindIIl Pst l BamHI XmalSmal

EcoRI HaellI

Sal lAccI

HincII

M13mp7�pUC71

THRACC

2METATG

3ILEATT

4THRACG

5ASNAAT

1ser

TCC

2pro

CCG

3aspGAT

4pro

CCG

5ser

TCG

6thr

ACC

7cys

TGC

8arg

AGG

9ser

TCGATG

10thr

ACG

11aspGAT

12pro

CCG

13gly

GGG

14asnAAT

Pst lBamHIEcoRI

6SERTCA

Sal lAccI

HincII

EcoRIBamHI

M13mpvector

lac i

lac z′

4

1

63

10

2

5 7 9 8

Figure 1.14.2 M13mp/pUC polylinkers. Sequence of polylinkers in the commonly used members of these two series ofvectors (Yanisch-Perron et al., 1985, and references therein). Amino acids that have been added to the lacZ gene productby insertion of the polylinker are shown in lower case letters. The bracket shows location of polylinkers on vector.

1.14.2

of the phage genome. The polylinker is embed-ded in-frame within an alpha fragment of thelacZ gene. M13mp derivatives form blueplaques on lawns of cells that contain the lacZomega fragment on plates with Xgal and IPTG.Double-stranded DNA is purified from phage-infected cells, cut within the polylinker withrestriction enzymes, and foreign DNA is ligatedto the cut vector. Insert-bearing phages formwhite plaques. Infected cells from the center ofwhite plaques are grown in liquid culture thencentrifuged to yield supernatants full of phageparticles containing a single strand of DNA.Phage particles are concentrated and pure sin-gle-stranded DNA is extracted from them andused in other procedures.

Two inadequacies of the M13mp phagessparked development of subsequent genera-tions of vectors. First, it is not always easy toobtain large amounts of double-stranded DNA.This is because DNA is obtained by lysinginfected cells, but infected cells grow moreslowly than uninfected cells, and there is noeasy selection for cells that retain the phage.Second, phages that contain insertions of morethan a few hundred nucleotides sometimes giverise to progeny phage in which some of theinserted DNA was deleted. This may be due tothe fact that cells infected with large phagesgrow even more slowly than cells infected withsmall phages, so that cells containing deletionderivatives of phages with large inserts have agrowth advantage.

In order to overcome these disadvantages,many vectors were developed (e.g., pEMBL)that contain a short stretch of DNA that includesthe phage origin of replication. These vectorsalso contain a pMB1-derived origin of replica-t ion, a β-lactamase (ampicillin-resistance)gene, and a polylinker embedded within analpha-complementing fragment of lacZ. Thevectors, which are much smaller than theM13mp phages, are introduced into the cell bytransformation; their continued presence can beensured by growth of the cells in ampicillin-containing medium. Since these vectors do notencode phage proteins required for DNA rep-lication, vector DNA replicates inside the cellsusing the pMB1 origin of replication. However,when cells containing these vectors are super-infected with wild-type helper phage, thephage origin of replication becomes active andsingle-stranded progeny phages are secretedinto the medium together with progeny helperphage. Contamination with helper phage usu-ally does not interfere with most applicationsbecause the helper does not contain genes en-

coding lacZ or ampicillin resistance, nor is itlikely to contain any piece of nucleic acid usedas a hybridization probe. Vectors developedeven more recently (e.g. , pUC118,pBluescribe) are designed to be used with aslightly improved helper phage and also ofteninclude SP6 or T7 phage polymerase promotersreading into the polylinker (see Fig. 1.5.3 andUNIT 1.15 for details).

LIFECYCLE OF FILAMENTOUSPHAGES

The filamentous phages (f1, M13, and fd)are single-stranded DNA phages that are male-specific—that is, they infect male E. coli strainsthat contain an F factor (F+, F′, or Hfr; see UNIT

1.4). These three phages are essentially inde-pendent isolates of the same phage, and differonly by a few nucleotide substitutions. Thephage particle is shaped like a long thin tube.Its coat is composed primarily of thousands ofmonomers of the gene 8 product. The genomeconsists of a single-stranded, circular DNAmolecule, 6407 nucleotides long, which runsdown the length of the tube. In addition tocoding for ten proteins, the genome contains anintergenic space (IG) between gene 4 and gene2. The IG contains origins for (+)-and (−)-strand DNA synthesis, a signal for packagingthe (+) strands into phage particles, and a tran-scription terminator. (A genetic map of thephage is shown in Figure 1.14.3.) Filamentousphages are useful cloning vectors because thereappears to be no size limit for packaging; iflonger genomes are generated, they are pack-aged into longer phage particles. The phageparticle contains ∼2700 monomers of the majorcoat protein, the product of gene 8, and severalminor coat proteins at the ends. One of theminor coat proteins, the product of gene 3,attaches to the receptor at the tip of the F pilusof the host E. coli. Upon binding, the pilus isthought to retract, bringing the phage in contactwith the bacterial cell surface. The coat proteinsare removed from the phage particle and in-serted into the bacterial cell membrane. Afteruncoating, the infecting circular single-stranded DNA is brought into the cytoplasm ina process that remains unclear.

Phage DNA replication takes place in threestages within the infected cell. In stage one(complementary strand synthesis), a comple-mentary (−) strand of phage DNA is synthe-sized. This synthesis converts the infecting sin-gle-stranded circular DNA, the (+) strand, intodouble-stranded replicative-form (RF) DNA.Synthesis of the (−) strand begins at the (−)-


1.14.3


strand origin (within the IG), and is carried outby the host E. coli replicative enzymes.

In the second stage of replication, the intra-cellular pool of RF DNA is increased by thesequential action of both replication origins.Rolling-circle type replication from the (+)-strand origin is followed by conversion of theprogeny single-stranded circles to doublestrands by replication starting at the (−)-strandorigin. The resulting RF DNA molecules areintermediates in DNA synthesis and transcrip-tion templates for the synthesis of the phage-encoded proteins. Transcription proceeds in thesame direction as (+)-strand rolling-circle rep-lication. It is suspected that transcription intothe replication fork, in the direction of the (−)strand, would inhibit replication, as proposedfor the replication of the E. coli chromosome(Brewer, 1988). Although this idea has notbeen rigorously tested, it is attractive becauseit provides an explanation for two commoncloning problems: why certain pieces of DNAare not clonable, and why, occasionally, onlyone of two possible orientations of an insertare sometimes recovered in a subcloning ex-periment.

The (+)-strand replication origin that directsrolling-circle replication has a bipartite struc-ture. It consists of an essential core origin re-gion (∼50 bp) and an adjacent A+T–rich “en-hancer” sequence (∼100 bp) which increasesreplication ∼100-fold. The core origin binds the

initiator protein (gene 2 protein) and the en-hancer binds the E. coli integration host factor(IHF). The polylinker cloning site in cloningvectors of the M13mp series disrupts the repli-cation enhancer sequence, but these vectorshave acquired compensatory mutations in gene2 that restore efficient replication. Conse-quently, virtually any sequence can be intro-duced into the polylinker without disruptingDNA replication.

The gene 2 protein is a multifunctional pro-tein that plays several roles in phage DNAreplication. It binds cooperatively to the (+)-strand origin in two steps, bends the originDNA, and introduces a specific nick in the (+)strand of RF DNA. The 3′-hydroxyl end of thenick serves as the primer for (+)-strand rolling-circle replication. (This activity makes the gene2 protein a useful enzyme for producinguniquely nicked DNA molecules in vitro.) Afternicking, the DNA molecules still need the gene2 protein for unwinding and replication. Finally,upon completion of a round of synthesis, gene 2protein cleaves and circularizes the displacedsingle strand (see UNIT 1.15).

The third stage of replication (single-strandproduction) occurs late in infection. This stageof DNA replication is asymmetric because the(−)-strand origin functions at a reduced levellate in the infection and therefore mainly (+)strands are produced. These single-strandedcircles, (+) strands, are packaged into phage

+ strand

– strand

ori 57

9 8

10

3

61

4

2

wild-typephage M13

NaeI (5615)BanII (5647)DraIII (5721)

Sau961 (5724)DrdI (5765)AvaI (5825)

SwaI (5943)

BpmI (6041)

AccI (6091)

HpaI (6407)

Alw44I (4743)

PacI (4136)

EarI (4080)

BspGI (3642)

BsrFI (3613)

RIeAI (3007)

DsaI (2763)

BamHI (2220)AlwNI (2192)

BseRI (2010)EciI (1964)

BsmI (1752)

NspBII (1633)

BspHI (1299)SnaBI (1270)

Bsp14071 (1021)

BsmFl (5091)MscI (5082)EaeI (5080)

6407 bp

Figure 1.14.3 Lifecycle of wild-type M13, described in this unit and in Rasched and Oberer (1986).


1.14.4

Introduction toVectors Derived

from FilamentousPhages

particles for export instead of being convertedinto RF DNA.

Ordinarily, infection with filamentousphages is not lethal and the host cells do notlyse, although their growth rate slows ∼2-fold.The reduced growth rate of the host cells ac-counts for the turbid plaques that the filamen-tous phage form on a lawn of sensitive cells.Surprisingly, several of the phage proteins havebeen found to be lethal to E. coli when ex-pressed from a plasmid with enhanced replica-tion (high copy number). Evidently, DNA rep-lication in the infected cell is tightly regulatedto prevent this lethality. The mechanismwhereby this regulation is achieved is not com-pletely understood, but the phage-encoded sin-gle-stranded DNA binding protein (gene 5 pro-tein) appears to be required for it. In the steadystate of infection, the RF DNA is maintainedas a plasmid with a copy number of ∼20 to 40,and phage are continuously exported at a rateof 100 to 200/hr. The infected state is quitestable with ∼1 cell in every 1000 cells becominguninfected each generation.

LITERATURE CITEDBeck, E. and Zink, B. 1981. Nucleotide sequence

and genome organization of filamentous bacte-riophage f1 and bacteriophage fd. Gene 16:35-38.

Brewer, B.J. 1988. When polymerases collide: Rep-lication and the transcriptional organization ofthe E. coli chromosome. Cell 53:679-686.

Dente, L., Cesareni, G., and Cortese, R. 1983.pEMBL: A new family of single-stranded plas-mids. Nucl. Acids Res. 11:1645-1655.

Greenstein, D. and Horiuchi, K. 1988. Integrationhost factor interacts with the DNA replicationenhancer of filamentous phage f1. Proc. Natl.Acad. Sci. U.S.A. 85:6262-6266.

Messing, J. 1983. New M13 vectors for cloning.Methods Enzymol. 101:20-78.

Messing, J., Gronenborn, B., Muller-Hill, B., andHofschneider, P.H. 1977. Single-strand filamen-tous DNA phage as a carrier for in vitro recom-bined DNA. Proc. Natl. Acad. Sci. U.S.A.74:3642-3646.

Rasched, I. and Oberer, E. 1986. Ff coliphages:Structural and functional relationships. Micro-biol. Rev. 50:401-427.

Yanisch-Perron, C., Vieira, J., and Messing, J. 1985.Improved M13 phage cloning vectors and hoststrains: Nucleotide sequences of the M13mp18and pUC19 vectors. Gene 33:103-119.

Zinder, N.D. and Boeke, J.D. 1982. The filamentousphage as vector for recombinant DNA: A review.Gene 19:1-10.

Zinder, N.D. and Horiuchi, K. 1985. Multiregula-tory element of filamentous bacteriophages. Mi-crobiol. Rev. 9:101-106.

Contributed by David Greenstein and Roger BrentMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts


1.14.5


UNIT 1.15 Preparing and Using M13-Derived VectorsCloning vectors derived from filamentous phage are extremely useful because they allowcloned DNA to be isolated as either single- or double-stranded DNA. This unit containsprotocols for preparing both forms of DNA and for characterizing inserts in M13-derivedvectors. A protocol is also presented for preparing single-stranded DNA from plasmidsusing superinfection with helper phage. This method is advantageous because it allowscloned DNA to be maintained in the form of a plasmid while permitting single-strandedDNA to be isolated for DNA sequencing.

BASICPROTOCOL

ISOLATING SINGLE M13-DERIVED VECTORSTo ensure homogeneity of stocks of native or insert-containing vectors, it is necessary tostart from a single infected cell, or from a single plaque. This is achieved by plating outserial dilutions of an original culture stock.

Materials

E. coli strain infected with M13-derived vector (e.g., JM101 infected withM13mp18)

2× TY medium (UNIT 1.1)20 mg/ml IPTG in H2O (stored in aliquots at −20°C)20 mg/ml Xgal in dimethylformamide (stored in aliquots at −20°C)45°C top agarose (UNIT 1.1)H plates, prewarmed to 37°C (UNIT 1.1)

5-ml Falcon tubes with caps or equivalent plastic tubes37°C incubator


1. Make a series of 1:10 dilutions of the infected strain or a phage stock in 2× TYmedium. Place 100 µl of each dilution into separate 5-ml Falcon tubes with caps.Label the tubes in order to keep better track of them.

The vector is usually stored in an infected host at −80°C in 20% glycerol.

The vector can also be stored as isolated DNA or as phage. DNA is introduced into cellsby transfection (UNIT 1.11); phage, by infection (described in steps 2 to 5 below). If the vectorcontains a plasmid origin and a drug-resistance marker, vector-containing cells can beisolated as single colonies on antibiotic-containing medium. If the vector forms viableplaques, vector-containing cells can be isolated by streaking single colonies from the tinypatch of cells in the center of the plaques.

The following steps assume the vector is an M13mp vector, which contains a stretch of DNAthat encodes the alpha fragment of β-galactosidase. These vectors make blue plaques oncells containing the β-galactosidase omega fragment (UNIT 1.4). Insertion of DNA into thepolylinker inactivates the gene encoding the alpha fragment and gives rise to vectors thathave colorless plaques.

2. To each tube add the following:

200 µl noninfected bacteria grown to saturation10 µl IPTG40 µl Xgal3 ml 45°C top agarose

It is convenient to prepare a larger batch of the first three ingredients beforehand.

3. Mix by rapidly inverting the tubes twice and pour on individual H plates prewarmedto 37°C.

Contributed by David Greenstein and Claude BesmondCurrent Protocols in Molecular Biology (1990) 1.15.1-1.15.8Copyright © 2000 by John Wiley & Sons, Inc.Supplement 9

1.15.1

Preparing and UsingM13-Derived

Vectors

4. Let the top agarose harden 10 min at room temperature and transfer the plates to a37°C incubator.

5. After overnight growth, save only the plates that contain less than ∼100 plaques.

BASICPROTOCOL

PREPARING SINGLE-STRANDED PHAGE DNAFROM M13-DERIVED VECTORS

Cells containing the vector and the insert are grown up in liquid medium. The cells arecollected by centrifugation, and single-stranded DNA is prepared from the phage particlesin the supernatant.

Materials

E. coli strain (male-type, e.g., JM105)M13-derived vector2× TY medium (UNIT 1.1)PEG solution (UNIT 1.7)TE buffer (APPENDIX 2)Buffered phenol (UNIT 2.1)3 M sodium acetate100% ethanol and cold 70% ethanol

Sterile toothpicks37°C shaking water bath

Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5)

1. Inoculate 50 ml of 2× TY medium with 0.5 ml of an overnight culture of E. coli. Theovernight should be grown from a single colony on a minimal plate that does notcontain proline. Dispense 2-ml aliquots in 10-ml culture tubes. Inoculate each tubewith a colorless phage plaque picked with a sterile toothpick.

Many male strains of E. coli used for propagation of single-stranded phage (e.g., JM101or JM105; Table 1.4.5) carry a deletion that removes the proA and proB genes, but alsocontain an F′ plasmid that contains the proA+ and proB+ genes. Picking a colony from aplate that lacks proline thus ensures that the cells contain the F′ plasmid, and that the phagewill be able to infect them.

2. Shake at 37°C for 5 to 8 hr.

3. Transfer 1.5 ml of the culture to a microcentrifuge tube. Centrifuge 5 min at roomtemperature.

4. Pour 1.25 ml of the supernatant into a fresh microcentrifuge tube.

To store the strain, resuspend the bacterial pellet by vortexing, adjust to 30% glycerol, andfreeze at −80°C.

5. Add 250 µl PEG solution. Mix and leave for 15 min at room temperature.

6. Centrifuge 5 min and discard the supernatant.

The phage pellet should be visible at this stage.

7. Centrifuge again for 5 min. Carefully remove all remaining traces of PEG using adrawn-out Pasteur pipet.

8. Add to the viral pellet 200 µl TE buffer. Resuspend the pellet completely, then add100 µl buffered phenol.


1.15.2


9. Vortex 1 min, let stand for 5 min, vortex again, and centrifuge 5 min.

10. Remove 175 µl from the upper aqueous phase; transfer to a new microcentrifuge tube.

11. Add 20 µl of 3 M sodium acetate and 400 µl of 100% ethanol.

12. Leave ≥1 hr at −20°C to precipitate the DNA, or 15 min in a dry ice/ethanol bath.

13. Centrifuge 5 min, pour off the supernatant, add 1 ml cold 70% ethanol, centrifugeagain, discard the supernatant, and dry briefly in a vacuum desiccator.

For some applications of the single-stranded DNA, such as oligonucleotide-directedmutagenesis (UNIT 8.1), it is advisable to use very clean DNA. For these applications,aspirate off the supernatants from this step using a drawn-out Pasteur pipet.

14. Dissolve the pellet in 25 µl TE buffer.

Yield can be as high as 10 �g DNA/ml culture.

15. Analyze 1 to 2 µl of the DNA solution by electrophoresis on a 1% agarose gel andstore the remainder at −20°C.

BASICPROTOCOL

PREPARING DOUBLE-STRANDED REPLICATIVE-FORM DNA

The disadvantage of many protocols for isolating double-stranded replicative-form (RF)DNA is that the DNA is isolated late in the infectious cycle, at a time when it is leastabundant. The following protocol involves isolation of double-stranded DNA fromchloramphenicol-treated cells. Addition of chloramphenicol at a low concentration (15µg/ml) soon after infection prevents the accumulation of the phage gene 5 protein, whichinhibits (−)-strand DNA synthesis. Enough gene 2 protein is synthesized in the first 15min to allow accumulation of RF DNA.

Materials

F+ or Hfr E. coli strain2× TY medium (UNIT 1.1)Recombinant phage20% glucose1 mg/ml chloramphenicol in ethanol, freshly preparedSorvall SS-34 rotor or equivalent

1. Inoculate 20 ml of 2× TY medium plus 0.1 ml of 20% glucose with uninfectedbacteria. Incubate in a 37°C shaking water bath until OD600 = 0.8 to 1.0.

2. Infect the cells with the recombinant phage at an MOI (see glossary in chapterintroduction) of 20 to 50. Incubate 15 min at 37°C.

3. Add 0.3 ml of 1 mg/ml chloramphenicol in ethanol (final concentration, 15 µg/ml)to the culture. Incubate the culture an additional 2 hr.

4. Centrifuge 10 min at 4000 × g (6000 rpm) to harvest the cells. Prepare double-stranded DNA using the usual procedures for isolation of plasmid DNA (see UNITS 1.6

& 1.7).


1.15.3


Vectors

BASICPROTOCOL

PREPARING SINGLE-STRANDED DNA FROM PLASMIDSUSING HELPER PHAGE

Cells containing plasmids with filamentous phage origins (usually the f1 origin) areinfected with helper phage. The helper phage provides the gene 2 protein that drives theplasmid into the f1 mode of replication. The gene 2 protein nicks the (+)-strand origin ofthe plasmid and initiates rolling-circle replication, resulting in the production of single-stranded circles of the plasmid DNA. The helper phage also provides the DNA packagingand export functions. Single strands of the plasmid are packaged into phage coats andsecreted into the supernatant. It is important to remember that only the (+) strand ispackaged efficiently. Therefore, only the DNA strand of the insert that is in the same5′→3′ orientation as the phage (+)-strand origin will be packaged.

Materials

F+ or Hfr E. coli strain (Table 1.4.5) containing a plasmid (pUC118, pBS,or equivalent; see commentary)

2× TY medium (UNIT 1.1)

37°C shaking water bath and 65°C water bathSorvall SS-34 rotor or equivalent

1. Grow cells in 2× TY (containing an appropriate antibiotic) at 37°C to an OD600 of0.1.

A fresh overnight culture started from a single colony can be diluted 1:50 and grown to anOD600 of 0.1. Generally, 1- to 5-ml cultures will yield enough DNA for sequencing (typicalyields are 0.2 to 1 �g plasmid single strands/ml culture).

2. Infect the cells at an MOI of 20. Some plasmids and bacterial strains seem to requireaddition of more phage (MOI of 50), while some give good yields with addition ofless phage (MOI 5 to 10).

When performing this protocol for the first time, it is helpful to try several different MOIs(1, 10, 20, and 50). Use the least amount of phage necessary to give a good yield of plasmidsingle strands because a portion of the input phage will always be recovered.

3. Grow the cells 4.5 hr at 37°C with vigorous shaking.

For convenience, the infected cells can be grown overnight and the supernatant collectedthe following day. In many cases, this longer growth period will result in contamination ofthe supernatant with chromosomal DNA from lysed bacteria.

4. Centrifuge 10 min at 4000 × g (6000 rpm). Collect the supernatant and heat at 65°Cfor 15 min to kill any residual bacteria. Prepare single-stranded DNA from thesupernatant (steps 4 to 13 of the second basic protocol, preparing single-strandedphage DNA from M13-derived vectors). Analyze the single-stranded DNA on anagarose gel (UNIT 2.5) with the helper phage serving as a control.


1.15.4


BASICPROTOCOL

INTRODUCTION OF PHAGE DNA INTO CELLS

Both double-stranded and single-stranded vector DNA can be introduced into CaCl2-treated competent bacteria by transfection, just as if the vector DNA molecules wereplasmids. Single-stranded DNA usually yields about ten times fewer transformants thanthe same amount of double-stranded molecules.

Special applications may require vector DNA grown on an F- strain. The fact that phageDNA can be introduced into cells by transformation also makes it possible to producephages from F- hosts. However, since phages produced by infection of these cells cannotinfect neighboring cells, phages introduced into F- cells should always contain a drug-re-sistance marker so that transformed cells can be selected on antibiotic-containing plates.

Methods of transformation described in UNIT 1.8 are used to introduce vector DNA intocells, with the following adaptations where appropriate.

1. If the vector contains a plasmid replicator and a drug-resistance gene, then simplyselect transformed cells on antibiotic-containing plates.

2. If the vector is able to form plaques, add 200 µl of noninfected cells at late log phase(OD600 = 0.6 to 0.8)—grown as in UNIT 1.2—to the plating-out mixture. Add 2.5 ml Htop agar (UNIT 1.1), plate the mixture as a lawn on an H plate (UNIT 1.1), and incubateat 37°C until plaques appear (as in first basic protocol).

BASICPROTOCOL

DETERMINING SIZE OF INSERTS INSINGLE-STRANDED VECTORS

This method allows a quick comparison of a large number of viral DNAs withoutpurification of the single-stranded DNA (Messing, 1983).

The size of inserts is estimated by comparing the mobility of DNA purified frominsert-containing phage to the mobility of DNA from phages lacking inserts or containinginserts of known size. This procedure allows one to compare the sizes of single-strandedcircular molecules that are several kilobases long; however, the resolution on agarose gelsusually does not allow detection of inserts that are <300 bp long.

Materials

2% sodium dodecyl sulfate (SDS)Loading buffer

1. Pipet 1.5 ml of an infected culture into a microcentrifuge tube.

2. Spin for 1 min. Remove 20 µl of the supernatant and mix with 1 µl of 2% SDS and3 µl loading buffer. Repeat this process with supernatants of cultures of vector phagewithout inserts, and of other phage which contain inserts of known size.

3. Run these samples on a 0.7% agarose gel and analyze as described in UNIT 2.5. Ifdesired, DNA can be transferred from gel to membrane and assayed with a radioactiveprobe, for example, to determine that the fragment has the right sequence as well asthe right size.


1.15.5


Vectors

SUPPORTPROTOCOL

DETERMINING INSERT ORIENTATION

Only one strand of the inserted DNA is contained in the phage’s (+) strand and made intophage particles. This means that phage DNAs containing identical inserts in oppositeorientations will hybridize with each other along the stretch of inserted DNA (see sketch1.15A). The structures thus formed have different electrophoretic mobility than twounassociated single-stranded viral DNAs. This property of single-stranded phage vectorscan be used to determine if two phages have an insert in opposite orientations (Howarthet al., 1981), which can be useful for sequencing both ends of a fragment of DNA.


5 M NaCl

1. Mix 20 µl of two supernatants with 1 µl of 2% SDS in a microcentrifuge tube. Inseparate tubes mix 40 µl of each individual supernatant with 2 µl of 2% SDS.

2. Add 2 µl of 5 M NaCl to each of the three tubes and incubate 30 min at 60°C.

3. Run all three samples on a 0.7% agarose gel and visualize bands by staining withethidium bromide as described in UNIT 2.5.

If many samples must be screened, it is possible to grow, lyse, and process recombinantinfected cells in wells of microtiter plates.

It is also possible to determine the relative orientation of inserts in purified preparationsof single-stranded DNA. To do this, mix 5 �l DNA from each of two independent plaqueswith 0.5 �l of 5 M NaCl. Incubate 30 min at 60°C, then analyze on an agarose gel asdescribed above.


Loading buffer50% glycerol0.2 M EDTA, pH 8.30.05% bromphenol blue

conversion tosingle-stranded

form

hybridization

hybridization of two phages containing the sameinsert in opposite directions

Sketch 1.15A


1.15.6


COMMENTARY

Background InformationIn M13 cloning vectors (Messing, 1983), a

portion of the E. coli lac operon which bears apolylinker (a stretch of DNA which containscontiguous recognition sites for many differentrestriction enzymes) is inserted in the inter-genic region (Messing et al., 1977). On anomega-fragment producing host, these vectorsform blue plaques, and vectors with insertsform white plaques.

A plaque formed by M13 vectors is a zoneof infected cells within a lawn of noninfectedcells. Infected cells grow more slowly thanuninfected cells. Plaques made by phage thatcontain inserts are usually smaller than thosemade by wild-type phage. This is becausephage DNA which contains inserts is replicatedmore slowly, and it takes longer for cells in-fected with those phages to produce progenyplaques. Many people prefer to store the recom-binant phages not as plaques but as purifiedsingle-stranded DNA.

Other single-stranded cloning vectors con-tain the origins of replication of phages like f1.These vectors usually also contain the pBR322plasmid origin of replication, a drug resistancecoding gene, and a polylinker inserted in frameinto the portion of the lacZ gene coding for thealpha peptide. Most of these vectors lack gene2, or the other phage genes necessary to formsingle-strand phage particles.

In cells these vectors are double-strandedand replicate using the pBR322 origin of rep-lication. However, when cells are infected withwild-type helper phages, the plasmids replicateusing the phage origin of replication, and sin-gle-stranded copies of the plasmid are encapsi-dated into phage filaments, which are thensecreted into the culture medium along withcopies of the helper. Contamination with helperphage does not interfere with most applicationsbecause the helper does not contain genes en-coding lacZ or ampicillin resistance, nor is itlikely to contain any piece of nucleic acid usedas a hybridization probe.

Perhaps the most common application of thesevectors is in sequencing using the dideoxymethod (UNIT 7.4). In this procedure, small frag-ments of DNA from a plasmid or lambda-derivedvector are subcloned into M13mp vectors. Vec-tors containing the inserts are identified by thefact that they form colorless plaques on a lawn ofomega-fragment producing cells growing onXgal + IPTG plates (see UNIT 1.4).

For further background information see

lifecycle of filamentous phages, UNIT 1.14, andkey references listed there.

Critical ParametersIsolating single M13-derived vectors.

Plates should be used immediately afterplaques have appeared. Phages should befreshly plated every time a single plaque is tobe picked for DNA amplification. Phages dif-fuse, and use of an old plate can result in crosscontamination. Cells to be infected and lawnsof infected cells must be grown at 37°C. If cellsare grown at temperatures below 34°C, sex pilido not form and phages cannot infect.

Preparing single-stranded phage DNAfrom M13-derived vectors. It is critical to re-move PEG from the preparations, since tracesof PEG inhibit the activity of many DNA po-lymerases. Contaminated templates can be ex-tracted with chloroform prior to ethanol pre-cipitation. However if step 7 (of the first basicprotocol) has been followed carefully, chloro-form extraction is not necessary.

Preparing replicative-form (RF) DNA.Typical yields of double-stranded DNA fromchloramphenicol-treated cells are 50 to 200 µgper 20-ml culture depending on how well theparticular recombinant phage grows. Too muchchloramphenicol will result in reduced yields,while too little will result in contamination withsingle-stranded DNA.

Sometimes after the cesium chloride gradientis equilibrated, a third band appears between theplasmid DNA band (lowest) and the genomicDNA band (highest). This is made of single-stranded DNA and should be left behind.

Preparing single-stranded DNA from plas-mids using helper phage. Start with a singlecolony of male E. coli strain that is harboring arecombinant plasmid containing a filamentousphage replication origin such as pUC118 (Vieraand Messing, 1987) or pBS (Stratagene).

The strain of helper phage used must beappropriate for the experiment. Several help-er phages such as IR-1 (Enea and Zinder,1982), R408 (Russell et al., 1986), M13K07(Veira and Messing, 1987), and VCSM13 (de-rived from M13K07; Stratagene) are available.The phages IR-1 and R408 are more stablethan VCSM13 and M13K07, but the latterphages contain a kanamycin-resistance genethat aids in selection. The helper phagesVCSM13 and R408 were designed to favor theproduction of plasmid single strands. VCSM13 has aphage (+)-strand replication origin with


1.15.7


Vectors

a defective replication “enhancer” sequence.Therefore, it doesn’t compete as well for thegene 2 protein and hence favors the productionof plasmid single strands. In contrast, the helperphage R408 has a defective packaging signal(see UNIT 1.14) causing the plasmid single strandsto be preferentially packaged into phage parti-cles. Regardless of the helper phage used, thesingle-stranded DNA prepared from the super-natant will contain some DNA from the helperphage. This is usually not a problem for DNAsequencing because a primer can be chosen thatis specific for the plasmid single strands.

Introduction of phage DNA into cells.Transformation of CaCl2-treated competentbacteria is carried out as described in UNIT 1.8. Ifthe vector forms plaques, it is not necessary tophenotypically express, and the cells can beplated out immediately following the heatshock step. Using F− competent cells is a com-mon mistake, since the recombinant phage willnot form plaques on a lawn of F− cells. How-ever, F− competent cells can be used if they arediluted with uninfected F+ cells following tran-sformation and plated together. In this case, theF− cells will produce phage that will makeplaques on the mixed lawn. As long as the F+

cells are in abundance, plaques will be seen.Determining size of inserts in single-

stranded vectors. Single-stranded DNA is ana-lyzed from a phage supernatant after extractionfrom the phage particle. It is important to vortexthe phage with the SDS to remove the coatproteins from the phage DNA. Failure to do sowill result in incomplete extraction and smear-ing of the DNA bands. If the culture supernatantcontains bacteria or chromosomal DNA fromlysed bacteria, the phage DNA will be contami-nated and other DNA bands will be visible. Ifthis is a recurrent problem, the position of theseextra bands can be determined by running anuninfected culture supernatant as a control.

Time ConsiderationsPlaques can be seen after ∼4 hr. The color

of plaques can be determined after a few morehours of incubation.

When preparing single-stranded phageDNA, incubation and processing samples areperformed the same day. The only limitingfactor is the number of samples to process atthe same time. If there are too many, it ispossible to store the supernatants (step 5, firstbasic protocol) at 4°C for 24 hr and then recen-trifuge before proceeding to the next step.

Preparing single-stranded DNA with helper

phage requires a shorter incubation period (4.5hr) after inoculation with the phage. PreparingRF DNA from chloramphenicol-treated cellscan be accomplished within one day after theincubation. An additional day may be requiredif the sample is to be purified on a CsCl gradi-ent.

Introducing phage DNA into cells, deter-mining insert size, and determining insert ori-entation each takes ∼2 hr.

Literature CitedEnea, V. and Zinder, N.D. 1982. Interference resis-

tant mutants of phage f1. Virology 122:222-226.

Howarth, A.J., Gardner, R.C., Messing, J., andSheperd, R.J. 1981. Nucleotide sequence of anaturally occurring deletion of a mutant of acauliflower mosaic virus. Virology 112:678.

Messing, J. 1983. New M13 vectors for cloning.Methods Enzymol. 101:20-78.

Messing, J., Gronenborn, B., Muller-Hill, B., andHofschneider, P.H. 1977. Single-strand filamen-tous DNA phage as a carrier for in vitro recom-bined DNA. Proc. Natl. Acad. Sci. U.S.A.74:3642-3646.

Russell, M., Kidd, S., and Kelley, M.R. 1986. Animproved filamentous helper phage for sequenc-ing single-stranded plasmid DNA. Gene 45:333-338.

Viera, J. and Messing, J. 1987. Production of single-stranded plasmid DNA. Methods Enzymol.153:3-11.

Key ReferencesDente, L., Sollazzo, M., Cesareni, G., and Cortese,

R. 1985. The pEMBL family of single-strandedvectors. In DNA cloning: A Practical Approach(D.M. Glover, ed.) pp. 101-108. IRL Press, Ox-ford.

Reviews biology of non-M13−derived single-stranded vectors.

Howarth et al. 1981. See above.

Describes techniques for determining insert orien-tation.

Messing, J. 1983. See above.

Maintenance, propagation, and titration of filamen-tous phage vectors are extensively described.

Contributed by David GreensteinMassachusetts General Hospital and Harvard Medical SchoolBoston, Massachusetts

Claude BesmondCentre de Recherche InsermParis, France


1.15.8


SECTION VSPECIALIZED TECHNIQUES

UNIT 1.16Recombineering: Genetic Engineering inBacteria Using Homologous Recombination

Over the past few years, in vivo technologies have emerged which, due to their efficiencyand simplicity, already complement and may one day replace standard genetic engineer-ing techniques. The bacterial chromosome and episomes can be engineered in vivo byhomologous recombination using PCR products and synthetic oligonucleotides (oligos)as substrates. This is possible because bacteriophage-encoded recombination functionsefficiently recombine sequences with homologies as short as 35 to 40 bases. This tech-nology, termed recombineering, allows DNA sequences to be inserted or deleted withoutregard to presence or location of restriction sites.

To perform recombineering, a bacterial strain expressing a bacteriophage recombinationsystem is required. The phage enzymes can be expressed from either their own promoteror from a heterologous regulated promoter. Expressing the genes from their endogenousphage promoter confers the advantage of tight regulation and coordinate expression,which results in higher recombination frequencies. This is an important advantage, sincein many cases high recombination frequencies will be essential to obtaining a desiredrecombinant. The authors of this unit routinely use a defective λ prophage located on theE. coli chromosome, and have recently transferred the critical elements of this prophageto a number of different plasmids (Thomason et al., 2005; also see Commentary). Inthis prophage system, the phage recombination functions are under control of the bac-teriophage λ temperature-sensitive cI857 repressor. At low temperatures (30◦ to 34◦C),the recombination genes are tightly repressed, but when the temperature of the bacterialculture is shifted to 42◦C, they are expressed at high levels from the λ pL promoter. In theplasmid construct of Datsenko and Wanner (2000), the recombination genes are locatedon a plasmid and expressed from the arabinose promoter. The Datsenko and Wannerplasmid and some of the authors’ plasmid constructs have temperature-sensitive originsof DNA replication. The plasmid-based systems have the advantage of mobility—theycan be transferred among different E. coli strains or to Salmonella typhimurium andpossibly other gram-negative bacteria. However, using the prophage system located onthe bacterial chromosome is more facile if the recombineering is targeted to a plas-mid. After induction of the recombination functions, the modifying DNA, either adouble-stranded (ds) PCR product or a synthetic single-stranded (ss) oligonucleotide(oligo), is introduced into the prophage-containing strain by electroporation. Recombi-nants are obtained either by selection or screening of the population of cells survivingelectroporation. Once the desired construct is obtained, the prophage can be removedby another recombination. Alternatively, engineered alleles on the chromosome can bemoved into a different host by P1 transduction. Plasmids with temperature-sensitivereplication origins can be lost from the recombinant strain by growth at the appropriatetemperature.

Preparation of electrocompetent cells that have expressed the recombineering functionsfrom the λ promoter and their transformation with double-stranded DNA (dsDNA) orsingle-stranded DNA (ssDNA) is the first procedure described in this unit (see BasicProtocol 1). Support Protocol 1 describes a two-step method of making genetic alterationswithout leaving any unwanted changes. In the latter protocol, the first of the two series

Contributed by Lynn Thomason, Donald L. Court, Mikail Bubunenko, Nina Costantino,Helen Wilson, Simanti Datta, and Amos OppenheimCurrent Protocols in Molecular Biology (2007) 1.16.1-1.16.24Copyright C© 2007 by John Wiley & Sons, Inc.


1.16.1

Supplement 78

GeneticEngineering inBacteria Using

HomologousRecombination

1.16.2


of steps is a recombineering reaction that replaces the sequence to be modified with anantibiotic-resistance cassette and a counter-selectable marker (e.g., sacB, which is toxicwhen cells are grown on medium containing sucrose; Gay et al., 1985). The secondseries of steps is a subsequent recombination that replaces the antibiotic cassette andthe counter-selectable marker with the desired genetic alteration. Moreover, the sameselection and counter-selection can be reused to make further modifications. SupportProtocol 2 describes a method for retrieving a genetic marker (cloning) from the E.coli chromosome or a co-electroporated DNA fragment and moving it onto a plasmid.Whereas the above protocols generally use selection to identify the recombinants, SupportProtocols 3 and 4 describe methods to screen for unselected mutations. Support Protocol 5details modifications necessary when targeting multicopy plasmids with recombineering.Basic Protocol 2 describes the removal of the defective prophage. Finally, AlternateProtocols 1 and 2 present methods for recombineering with an intact prophage and forintroducing mutations onto bacteriophage λ, respectively.

STRATEGIC PLANNING

Before attempting to modify the E. coli chromosome or a plasmid, the DNA sequence ofthe desired final construct should be determined. A DNA-analysis computer program suchas Gene Construction Kit (GCK; Textco Software; http://www.textco.com/) or Vector NTI(Invitrogen) is invaluable for this task. Having the sequence of both the original genomearrangement and the designed final construct as electronic files facilitates the design ofoligonucleotides to be used as primers for PCR or as ssDNA recombination substratesthemselves. The computer-determined sequences also allow rapid design of primers toanalyze and verify the potential recombinants. One must be aware of gene-regulationissues when designing the constructs. Bringing in a promoter with an antibiotic cassettecan help in establishing drug resistance; however, transcription from this promoter canextend beyond the drug marker and affect distal genes. The authors of this unit havedesigned several drug cassettes with their promoter, open reading frame, and transcriptionterminator region, as described in Yu et al. (2000); primers for amplification are listedin Table 1.16.2. One must also be careful of possible polarity effects and avoid creatingunwanted fusion proteins when generating recombinants.

The DNA substrate used to generate the recombinants depends on the desired change.For a sizeable insertion, such as a drug cassette, a PCR product is generated that contains40 to 50 base pairs of flanking homology to the chromosomal or plasmid target at eachend. This homology is provided at the 5′ end of each synthetic primer. Following thisregion of chromosomal homology, ∼20 bases of homology to the drug cassette providesthe primers to amplify the cassette sequence. Thus, two primers are designed that willeach be 60 to 70 nucleotides (nt) long: the 5′ ends provide homology to the targetedregion and the 3′ ends provide homology to the cassette (see Fig. 1.16.1). Careful primerdesign is crucial (see above). The efficiency of recombineering with dsDNA can approach0.1% (Yu et al., 2000). If deletions, small substitutions, or base changes are desired, asynthetic single-stranded oligo of ∼70 to 100 nt can be used. The oligo should have35 to 40 nt of complete homology flanking the alteration. Recombineering with ssDNAin wild-type E. coli containing the defective λ prophage gives efficiencies approaching1% (Ellis et al., 2001), and if host mismatch repair is inactivated, either by mutation orby using an oligo that creates a C-C mismatch, a 20% to 25% recombination frequencyis achievable (Costantino and Court, 2003). This extremely high frequency means that,for oligo recombination, it is possible to create recombinants without a selection andfind them by screening. Since an oligonucleotide corresponding to the lagging strand ofDNA replication is some 20-fold more efficient than its complement, it is worthwhile todetermine the direction of replication through one’s region of interest and use the oligothat corresponds to the lagging strand (Costantino and Court, 2003).


1.16.3


Figure 1.16.1 Targeting of an antibiotic cassette. Two primers with 5′ homology to the target areused to PCR amplify the antibiotic cassette. The PCR product is introduced by electroporation intocells induced for the Red recombineering functions. The Red functions catalyze the insertion ofthe cassette at the target site, which may be on the bacterial chromosome or on a plasmid.

It is also possible to rescue or clone gene(s) from the bacterial chromosome onto aplasmid. To do this, create a linear PCR product of the plasmid using primers withhomology flanking the target sequence, designed so that the sequence will be incorporatedonto the circular plasmid in the appropriate orientation. The PCR-amplified linear plasmidwill require an origin of DNA replication and a selective marker.

Multicopy plasmids can also be modified with recombineering, and changes occur at thesame frequency as modifications of the E. coli chromosome (Thomason et al., 2007). Thismeans that point mutations can be made at a frequency of ∼5% to 30% in the absence ofmismatch repair. The frequency of insertion or removal of large DNA segments is muchlower, however, and isolation of recombinant species will be problematic in the absence ofa selection or screen. Designing the loss of a unique restriction site into the recombinantplasmid permits enrichment of the recombinant class by allowing destruction of theunmodified parental population by restriction digestion.

Some modification of Basic Protocol 1 is needed when targeting plasmids with recom-bineering, due to the fact that they exist in multiple copies in the cell. Different plasmidsvary widely in their intracellular copy number, ranging from low copy vectors with onlya few molecules per cell, such as pSC101, to pUC-based plasmids that routinely have∼500 copies per cell. This means that it is generally impossible to modify all copiesof the episome of interest during a recombineering reaction, and thus cells containing



1.16.4


recombinant plasmids will also contain unmodified parental plasmid. The recombinantclass of molecules can be separated from the unmodified class by plasmid DNA isolationand retransformation at a DNA concentration of less than one molecule per cell, whichcreates pure clonal populations of the recombinant plasmid species. Parental plasmidscan also be eliminated by restriction digestion as mentioned above.

A complexity arising when plasmids are modified with recombineering is that circularmultimeric plasmid species are formed during the recombination reaction. In the absenceof linear substrate DNA, expression of the Red proteins does not cause plasmids tomultimerize under the conditions given here (Thomason et al., 2007), but when linearsingle-stranded or double-stranded substrate DNA is added and modification of the targetsequence occurs, circular multimeric species are formed. Multimer formation is thus ahallmark of a recombinant plasmid molecule (and could theoretically be used to identifyrecombinant species). It is important to begin recombineering with a monomer plasmidspecies and to do the reaction in a recA mutant bacterial strain defective in homologousrecombination. Using a host defective in RecA-mediated recombination eliminates oneroute to plasmid-by-plasmid recombination.

A decision should be made as to whether the plasmid will be introduced into the Red-expressing cells by co-electroporation with the linear DNA after the recombinationfunctions are induced, or whether the plasmid should already be established in thebacteria. Co-electroporation offers the advantage that it helps control plasmid copynumber at the time of recombineering and minimizes opportunities for plasmid multimerformation. However, for very large plasmids of low copy number, co-electroporationmay not be feasible.

BASICPROTOCOL 1

MAKING ELECTROCOMPETENT CELLS AND TRANSFORMING WITHLINEAR DNA

This basic protocol describes making electrocompetent cells that are preinduced for therecombination functions and transforming them with the appropriate DNA to createthe desired genetic change. As noted in Strategic Planning, the phage recombinationfunctions are repressed by the phage λ temperature-sensitive cI857 repressor, so thatthey are not expressed when the cells are grown at low temperature (30◦ to 34◦C) butare highly expressed when the culture is shifted to 42◦C. See Commentary for additionalconsiderations before executing the procedure.

Materials

Purified PCR product or oligonuclotide primers with ∼40 to 50 bases of flankinghomology on either side of desired change (also see UNIT 15.1)

Bacterial strain expressing the defective lambdoid prophage recombination systemλ Red (Table 1.16.1; strains are available from the Court Laboratory;[email protected])

LB medium and plates (UNIT 1.1), without antibioticMedium lacking carbon source: M9 medium (UNIT 1.1) or 1× TM buffer (APPENDIX 2)Selective plates (UNIT 1.1)—minimal plates if selecting for prototrophy or rich plates

containing antibiotic (depending on drug cassette used):30 µg/ml ampicillin30 µg/ml kanamycin10 µg/ml chloramphenicol12.5 µg/ml tetracycline50 µg/ml spectinomycin

30◦ to 32◦C incubator32◦ and 42◦C shaking water baths


1.16.5


Table 1.16.1 Bacterial Strains Commonly Used for Recombineering

Strain Genotype

DY329 W3110 �lacU169 nadA::Tn10 gal490 pgl�8 λcI857�(cro bioA) (TetR)

DY330 W3110 �lacU169gal490 pgl�8 λcI857 �(cro-bioA)

DY331 W3110 �lacU169 srlA::Tn10 �recA gal490 pgl�8 λcI857�(cro-bioA) (TetR)

DY378 W3110 λcI857 �(cro-bioA)

DY380a mcrA �(mrr-hsdRMS-mcrBC) φ80dlacZ�M15 lacX74 deoR recA1endA1 araD139 �(ara, leu)7697 galU gal490 pgl�8 rpsL nupGλ(cI857ind1) �{(cro-bioA)<>tetRA} (TetR)

DY441 W3110 gal490 pgl�8 λcI857 �(cro-bioA) int<>cat-sacB

HME5 W3110 �lacU169 λcI857 �(cro-bioA)

HME45b W3110 gal490 pgl�8 λcI857 �(cro-bioA)

HME63 W3110 �lacU169 λcI857 �(cro-bioA) galKam mutS<>amp

HME64 W3110 �lacU169 λcI857 �(cro-bioA) galKam uvrD<>kanaDH10B derivative (Invitrogen).bGives less background on low concentrations of chloramphenicol than DY378.

125- and 250-ml Erlenmeyer flasks, preferably baffledRefrigerated low-speed centrifuge with Sorvall SA-600 rotor (or equivalent)35- to 50-ml plastic centrifuge tubes0.1-cm electroporation cuvettes (Bio-Rad), chilledElectroporator (e.g., Bio-Rad E. coli Pulser)

Additional reagents and equipment for PCR (UNIT 15.1), agarose gel electrophoresisof DNA (UNITS 2.5A & 2.6), purification of DNA by ethanol precipitation (UNIT 2.1A;optional; commercially available PCR cleanup kit may be substituted),electroporation (UNIT 1.8), isolation of bacterial colonies by streaking (UNIT 1.3),restriction enzyme digestion (UNIT 3.1), and DNA sequencing (Chapter 7)

Prepare DNA for transformation1. Design and procure the oligos to use for PCR-mediated generation of a dsDNA

product, or for use in single-stranded oligo engineering.

The sequences of the primers used to amplify the common drug cassettes are listed inTable 1.16.2. Remember to add the homologous targeting sequence to the 5′ ends of theoligos.

UNIT 15.1 describes general considerations for primer design.

2. Make the PCR product (UNIT 15.1), examine it by agarose gel electrophoresis (UNIT 2.5A),and gel purify by isolating the desired band (UNIT 2.6) if unwanted products areobtained.

If the DNA is gel purified, avoid exposing it to ultraviolet light, which will damage it andresult in lower recombination frequencies.

3. Clean up the PCR product by ethanol precipitation (UNIT 2.1A) or using a commerciallyavailable kit to remove salt.

If a plasmid template is used to construct a PCR-amplified drug cassette, any intact circu-lar plasmid remaining will transform the cells efficiently and give unwanted background.This background can be minimized by using a linear plasmid template for the PCR andby digesting the completed PCR reaction with DpnI before using it for electroporation.Always include a control reaction of uninduced cells transformed with the PCR product,to give a measure of any unwanted intact plasmid background.



1.16.6


Table 1.16.2 PCR Primers and Suggested Source of Template for Amplifying Drug Cassettes(from Yu et al., 2000)

Gene Source Primer sequence

Ampicillin pBluescript SK(+)(Stratagene)

5′ CATTCAAATATGTATCCGCTC5′ AGAGTTGGTAGCTCTTGATC

Tetracycline Tn10 5′ CAAGAGGGTCATTATATTTCG5′ ACTCGACATCTTGGTTACCG

Chloramphenicol pPCR-Script Cam(Stratagene)

5′ TGTGACGGAAGATCACTTCG5′ ACCAGCAATAGACATAAGCG

Kanamycin Tn5 5′ TATGGACAGCAAGCGAACCG5′ TCAGAAGAACTCGTCAAGAAG

Spectinomycin DH5αPRO (Clontech) 5′ ACCGTGGAAACGGATGAAGGC5′ AGGGCTTATTATGCACGCTTAA

Prepare bacterial cultures4. Inoculate the suitable bacterial strain (Table 1.16.1) from frozen glycerol stock or a

single colony into 3 to 5 ml LB medium. Shake at 30◦ to 32◦C overnight.

Most of the authors’ strains containing the defective lambdoid prophage are W3110derivatives; however, the prophage can be moved into other backgrounds. SeeCommentary for details. Plasmids expressing the recombination functions can be putinto any strain of choice.

Either 30◦ or 32◦C is acceptable for the low temperature throughout the procedure, sinceeither temperature allows good repression by the cI857 repressor. The cultures will growmore rapidly at 32◦C.

5. Add ∼0.5 ml of the overnight culture to 35 ml of LB medium in a 250-ml (baffled)Erlenmeyer flask.

This is a 70-fold dilution. One must make sure that one’s dilution is at least 50-fold.Higher dilutions will also work, but the cells will take longer to grow to the appropriatedensity. If targeting the recombineering to a plasmid, or expressing from a plasmid, addantibiotic as appropriate to maintain selection during growth. If an alternate method ofinducing the recombination functions is used (i.e., addition of arabinose), the inducershould be added to the medium. In this case remember to include an additional flaskcontaining an uninduced culture as a negative control. Expression of the λ Red functionsfrom the plasmids of Datsenko and Wanner (2000) is enhanced by use of 10 mM arabinose(for Ara+ strains).

6. Place the flask in the 32◦C shaking water bath and grow cells at 32◦C with shakingfor ∼2 hr.

The time will vary with different strains and dilutions. The cells are ready when the A600

is between 0.4 and 0.6. It is important not to over-grow the cells, since stationary phasecells do not express the recombination functions well.

Induce recombination functions7. Transfer half the culture to a 125-ml (baffled) Erlenmeyer flask and place that flask

in the 42◦C water bath. Shake 15 min at 220 rpm to induce. Leave the remainderof the culture at 32◦C; this will be used as the uninduced control that lacks recom-bination activity. While the cells are inducing, fill an ice bucket with an ice-waterslurry.


1.16.7


8. Immediately after inducing for 15 min at 42◦C, rapidly cool the flask in the ice-water slurry with gentle swirling. Leave on ice for ≥5 min. Follow the same coolingprotocol with the uninduced 32◦C culture. While the cells are on ice, precool thecentrifuge to 4◦C and chill the necessary number of 35- to 50-ml plastic centrifugetubes, labeled for induced and uninduced cells.

The temperature shift is unnecessary when a chemical inducer like arabinose is used.

Make electrocompetent cells9. Transfer both the induced and uninduced cultures to the appropriately labeled chilled

35- to 50-ml centrifuge tubes. Centrifuge 7 min at 4600 × g (6700 rpm in a SorvallSA-600 rotor), 4◦C. Aspirate or pour off supernatant.

10. Add 1 ml ice-cold distilled water to the cell pellet in the bottom of each tube andgently resuspend cells with a large pipet tip (do not vortex). Add another 30 mlice-cold distilled water to each tube, seal, and gently invert to mix, again withoutvortexing. Centrifuge tubes again as in step 9.

All subsequent resuspensions of cells through step 16 should be done gently and withoutvortexing. Preparation of the cells for electroporation washes out any added chemicalinducing agent.

11. Decant the 30-ml supernatant very carefully from the soft pellet in each tube andresuspend each cell pellet in 1 ml ice-cold distilled water.

Remove tubes from the centrifuge promptly. The pellet is very soft and care should betaken not to dislodge it, especially when processing multiple tubes.

12. Transfer resuspended cells to microcentrifuge tubes. Microcentrifuge 30 to 60 sec atmaximum speed, 4◦C. Carefully aspirate supernatant. In each of the tubes, resuspendthe cell pellet in 200 µl cold distilled water, which will provide enough material forfour or five electroporations.

For routine procedures when optimal recombination frequency is not necessary, e.g., whenselection is used to find recombinants, electrocompetent cells can be stored at −80◦Cafter resuspending the cell pellet in 15% (v/v) glycerol. For highest efficiency, use freshlyprocessed cells.

Introduce DNA by electroporation13. Chill the desired number of 0.1-cm electroporation cuvettes on ice. Turn on the

electroporator and set to 1.80 kV.

Brands of cuvettes and electroporator other than Bio-Rad may work, but have not beentested in the authors’ laboratory. The larger 0.2-cm cuvettes may require different elec-troporation conditions (consult electroporator instruction manual) and standardizationto obtain optimal recombination frequencies.

14. In microcentrifuge tubes on ice, mix 100 to 150 ng of salt-free PCR fragment (fromstep 3) or 10 to 100 ng of single-stranded oligonucleotide with 50 to 100 µl of thesuspension of induced or uninduced cells (from step 12). Do the mixing and subse-quent electroporation rapidly; do not leave the DNA-cell mixes on ice for extendedperiods. Be sure to include the following electroporation reactions and controls:

a. Induced cells plus DNA.

This is the culture that should yield the designed recombinants.

b. Induced cells without DNA.

This is a control to identify contamination, determine the reversion frequency, andobtain some idea of the efficiency of the selection.



1.16.8


c. Uninduced cells plus DNA.

This control tells whether there is some contaminating factor in the DNA that iscontributing to the selected colonies (for example, intact plasmid template from thePCR reaction will give rise to drug-resistant colonies here).

15. Introduce the DNA into the cells by electroporation (UNIT 1.8).

The time constant should be greater than 5 msec for optimal results. Low time constantsindicate problems with the cells, the DNA, or even the equipment.

16. Immediately after electroporation, add 1 ml LB medium to the cuvette using amicropipettor with a 1000-µl pipet tip. If transforming with a drug cassette, transferthe electroporation mix to sterile culture tubes and incubate the tubes with shakingat 30◦ to 34◦C for 1 to 2 hr to allow expression of the antibiotic resistance gene.

Even if not selecting for drug resistance, it is still recommended that the cells outgrow torecover from the shock of electroporation. It has been observed in the authors’ laboratorythat omitting the outgrowth reduces the cell viability ∼10-fold.

An alternative, and in the author’s experience more reliable, method for outgrowth is tospread appropriate dilutions of cells (see step 18 below) on a sterile 82-mm-diameternitrocellulose filter atop a rich (LB) plate. Incubate this plate for 3 hr at 30◦ to 34◦C.After the incubation, transfer the filter to the appropriate selective drug plate usingsterile forceps. This method is preferable because the cells are less dense and moreefficient outgrowth is achieved.

Determine cell titers17. Make serial 1:10 dilutions of the electroporation mix through 10−6 using M9 medium

or 1× TM buffer, dispensing 0.9 ml M9 or TM and 0.1 ml of the cell suspension pertube.

The dilutions can be made in rich medium if a selection for antibiotic resistance is applied.

18. To determine total viable cell count, spread 100 µl of 10−5 and 10−6 dilutions on LBplates (rich plates without drug). Incubate the plates at 30◦ to 34◦C for 1 to 2 days,depending on the growth requirements of the recipient strain.

19. To determine recombinant cell count, plate cells on selective plates as follows de-pending on the anticipated recombinant frequency.

a. If efficient recombination is expected, spread both 10 and 100 µl of the 10−1 and10−2 dilutions.

b. If low numbers of recombinants are expected, spread 100 µl each of a 1:5 and1:10 dilution.

The authors routinely obtain ten-fold or higher recombinant yields with the prophagesystem than with the Datsenko and Wanner plasmids.

For the no-DNA and uninduced controls, plate 200 µl directly on selective plates.

Since targeting to the chromosome results in a lower copy number of the drug cassettethan is present with a multicopy plasmid, antibiotic concentrations must be adjustedaccordingly. The authors routinely use the drug concentrations recommended above forchromosomal constructs. Minimal plates are used for selection based on prototrophy.

20. Incubate plates at the appropriate temperature (30◦ to 34◦C).

At 30◦C, colonies may take two days to come up on LB plates and 3 to 4 days on minimalplates. Candidates should be purified by streaking for single colonies and retested for theappropriate phenotype.


1.16.9


Analyze recombinants21. Once recombinant clones are identified, confirm the presence of the desired mu-

tation(s) by PCR analysis (UNIT 15.1) followed by DNA sequencing (Chapter 7) orrestriction digestion analysis (UNIT 3.1), if appropriate.

The design of the PCR primers depends on the changes made. For an antibiotic cassette orother insertion, the primer pair used to amplify the insertion can also be used to confirmits presence. These primers will not determine whether the cassette has integrated atthe desired location, however. The recombinant junctions can be confirmed with thehelp of two additional primers (all four should have compatible annealing temperatures)pointing outwards from the cassette; use one primer flanking the cassette and one internalcassette primer to amplify the unique junctions created by the recombination reaction. Aprimer pair that hybridizes to the external flanking sequences on each side rather than theinsertion itself can also be used to demonstrate loss of the target sequences and presenceof the insertion. Unwanted mutations can be introduced by heterologies (variations) inthe synthetic primer population (Oppenheim et al., 2004); therefore, it is important toconfirm the final construct by sequence analysis, especially the regions derived from theoriginal primers.

SUPPORTPROTOCOL 1

MANIPULATING cat-sacB FOR COUNTER-SELECTION AND GENEREPLACEMENT

This protocol describes a two-step method to create precise genetic changes withoutotherwise altering the DNA sequence. First, cat-sacB (or another counter-selectablecassette) is placed on the DNA; this is then replaced with the desired alteration in asecond recombineering event. The final construct will not have a drug marker.


Template for amplification of cat-sacB: bacterial strain DY441 (DY329 with acat-sacB insertion on the E. coli chromosome) or the plasmid pEL04 (Lee et al.,2001). pEL04 has previously been called both pK04 and pcat-sacB. BothDY441 and pEL04 are available from the Court Laboratory; [email protected].

SpeI and DpnI restriction endonucleases (UNIT 3.1)Primer L sacB: 5′-homology sequence-ATC AAA GGG AAA ACT GTC CAT

AT-3′

Primer R cat: 5′-homology sequence-TGT GAC GGA AGA TCA CTT CG-3′

Invitrogen Platinum High Fidelity enzymeLB-Cm plates: LB plates (UNIT 1.1) containing 10 µg/ml chloramphenicolM63 minimal glycerol plates with sucrose (see recipe) or LB plates (UNIT 1.1)

lacking NaCl but containing 6% (w/v) sucroseMedium lacking carbon source: e.g., M9 medium (UNIT 1.1)Thermal cycler (MJ Research PTC-100)

Amplify the cat-sacB element for recombineering1. If using pEL04, completely cleave pcat-sacB with SpeI.

This step is unnecessary if the chromosomal insertion of cat-sacB is used as template forPCR. Failure to digest when the plasmid is used as template will give a high backgroundof intact plasmid transformants.

2. Amplify cat-sacB, either from the cleaved pEL04 or from DY441 with the InvitrogenPlatinum High Fidelity enzyme and an MJ Research thermal cycler, using 50 pmolof each primer and the following cycling program:



1.16.10


1 cycle: 2 min 94◦C (denaturation)9 cycles: 15 sec 94◦C (denaturation)

30 sec 55◦C (annealing)3.5 min 68◦C (extension)

19 cycles: 15 sec 94◦C (denaturation)30 sec 55◦C (annealing)3.5 min(adding 5 sec/cycle)

68◦C (extension)1 cycle: 7 min 68◦C (extension)1 cycle: indefinite 4◦C (hold)

The PCR primers used to amplify the cat-sacB element are at the 3′ end of chimericprimers that include 5′ segments of bacterial target homology ∼40 to 50 nt in length.Hence each primer is 60 to 70 nt long.

Since the PCR product is greater than 3 kb, it can be difficult to amplify. The aboveconditions have been used successfully in the authors’ laboratory.

3. If pEL04 was used as template, digest the completed PCR reaction with DpnI tospecifically eliminate the methylated plasmid template. Purify the PCR product toremove salt (UNIT 2.1A).

Perform electroporation and recombination to insert cat-sacB at the desired location4. Insert the cat-sacB cassette into the chromosome as described above (see Basic

Protocol 1, steps 4 to 21; also see Background Information for important tips) usingthe following techniques specific for the cat-sacB cassette.

a. Select chloramphenicol-resistant (CmR) colonies and purify on LB-Cm plates toisolate single colonies.

b. Test several CmR isolates for sensitivity to sucrose, either on minimal glycerolplates containing 5% sucrose or on LB plates lacking NaCl but containing 6% su-crose (Blomfield et al., 1991). Use the parental transformation strain as a sucrose-resistant control. Determine that the insertion is correct before proceeding withthe next step of the procedure.

The sucrose-resistant parent serves as a control for growth in the presence of sucrose.Sucrose sensitivity needs to be tested because the PCR process generates mutationsthat inactivate sacB in a fraction of the clones.

Perform electroporation and recombination to replace cat-sacB with chosen allele5. Use a confirmed CmR/sucrose-sensitive candidate from step 4 as the starting bacterial

strain for a second round of recombineering by carrying out Basic Protocol 1, butsuspend the electroporated cells at step 16 in a final volume of 10 ml instead of 1 mlof LB medium, and incubate with aeration (applied via shaking in a shaking waterbath) at 30◦ to 32◦C for 4 hr to overnight for outgrowth following electroporation.

The higher dilution promotes better cell recovery and allows complete segregation ofrecombinant chromosomes that no longer carry the cat-sacB cassette from nonrecom-binant sister chromosomes that still contain it. If outgrowth is inadequate and sisterchromosomes are not fully segregated, the presence of the cassette on one chromosomewill confer sucrose sensitivity to the entire cell, thus preventing recovery of recombinants.This is generally true in counter-selection experiments and illustrates a common problemencountered with them.

6. Centrifuge cells 7 min at 4600 × g, 4◦C. Remove supernatant, then wash the cellstwice, each time by resuspending in l ml minimal medium lacking a carbon source,such as M9, centrifuging again as before, and removing the supernatant. Resuspendand dilute the cells for plating, and spread appropriate dilutions of the cells on either


1.16.11


LB sucrose plates or minimal glycerol plates (if the defective prophage was movedto another strain, the metabolic requirements of that strain must be considered). Inparallel, be sure to include a control of electroporated cells to which no PCR productwas added, to determine the frequency of spontaneous sucrose-resistant mutants;these spontaneous mutants will retain the CmR cassette.

The frequency of spontaneous sucrose-resistant cells is normally ∼1 in 104. Thus, inthe recombination experiment, the sucrose-resistant colonies that arise are of two types,spontaneous mutants like those found in the control, and deletions caused by replacingcat-sacB by recombination with the PCR product. The frequency of the latter is optimally10- to 100-fold greater than that of spontaneous mutation.

7. Purify ∼10 sucrose-resistant colonies by streaking to isolate single colonies (UNIT 1.3)and then test for chloramphenicol sensitivity (CmS)—spontaneous mutants will beCmR while the recombinants will be CmS. Screen the CmS/sucrose-resistant coloniesby PCR (see Basic Protocol 1, step 21), then sequence to confirm the presence of thedesired change.

Screen a minimum of ten colonies. If high-efficiency recombination is not achieved, morecolonies will need to be screened. Note that the frequency of spontaneous mutants remainsrelatively constant and provides an internal control for determining the efficiency of therecombination.

SUPPORTPROTOCOL 2

RETRIEVAL OF ALLELES ONTO A PLASMID BY GAP REPAIR

Often it is desirable to retrieve a DNA sequence from the bacterial chromosome, eitherto clone and amplify a gene, to express a gene under a given promoter, or to create agene or operon fusion. To do this with recombineering, a PCR product with homology tothe target at the ends is made from a linearized plasmid DNA and introduced into cellsexpressing the Red system. This homology will allow recombination with the sequenceto be retrieved, yielding a circular plasmid containing the sequence. It is important tolinearize the plasmid DNA used as template. This retrieval method works with ColEIand p15A (pACYC) replicons, but not with pSC101 (Lee et al., 2001).

If the desired gene is not present on the chromosome, it can be provided as a PCRproduct and introduced into the cells along with the PCR-amplified vector DNA by co-electroporation. Always remember to provide flanking homology so that the plasmid canrecombine with the additional PCR product (it is easier to provide the homology on theshort product to be cloned rather than on the vector).


Plasmid onto which sequence of choice is to be rescuedRestriction enzyme(s) (UNIT 3.1) that do not cut within plasmid region to be

amplifiedSynthetic chimeric primers providing homology to sequence flanking gene of

choice and to the plasmid sequence to be amplified

Amplify linear plasmid PCR product with homology to the target1. Design primers with homology that flanks the desired target.

Drawing a sketch of the plasmid as a gapped circle interacting with the target sequencewill help one visualize the recombination reaction (see Fig. 1.16.2), since the plasmidtemplate has the linear ends pointing toward each other. Both of the primers will have∼50 nt of bacterial sequence homology at the 5′ ends linked to 3′ plasmid sequence.It is not necessary to amplify the entire plasmid. Any portion can be amplified as longas the minimal requirements of a selectable marker and an origin of DNA replicationare met. If the DNA to be retrieved is adjacent to an antibiotic resistance gene, only aplasmid replication origin need be amplified; the origin can be used to retrieve both thedesired sequence and the nearby drug marker. Avoid having other regions of the plasmid



1.16.12


Figure 1.16.2 Cloning genes by gap repair of a plasmid. A linear plasmid with flanking homologyto the target at the ends (indicated by dark arrows) is generated by PCR. The plasmid is introducedby electroporation into cells expressing the Red functions, which catalyze recombination of thevector with the target site, resulting in incorporation of the gene onto the plasmid.

that are homologous to the bacterial chromosome (e.g., lac); these can lead to unwantedrearrangements.

2. To minimize background, digest the plasmid with one or more restriction enzyme(s)that do not cut within the region to be amplified. Amplify the linear plasmid by PCRusing the primers (reaction conditions will need to be established empirically).

The amplified product will be a linear gapped plasmid with flanking homology to eitherside of the allele to be rescued from the chromosome.

Use the least amount of plasmid DNA possible for the PCR template, to minimize the back-ground of false positives. Digestion of the completed PCR reaction mix with DpnI will helpremove the template plasmid. Purify the PCR product to remove salt before proceeding.

Transform induced cells with the linear plasmid and select recombinants3. Introduce the linear plasmid PCR product into the strain (see Basic Protocol 1, steps

4 to 21). If necessary, also add the PCR product to be co-electroporated. Selectfor the marker on the plasmid, and transform the uninduced cells with the linearplasmid PCR product, to determine the background of intact plasmid present. Purifycandidate colonies and screen them with PCR. Isolate the recombinant plasmids anduse them to retransform a standard cloning strain such as DH5α or XL2 Blue, togenerate pure clones.

Transformation into a recA mutant host ensures that the newly engineered plasmid doesnot undergo additional rearrangement.

If the DNA of interest is linked to a drug marker and it is retrieved with a replication originlacking a drug marker, dilute the electroporation mix into 10 ml LB medium and grow theculture overnight nonselectively. The next day, isolate plasmid DNA and transform intoa high-efficiency cloning strain, selecting for the drug resistance of the rescued marker.The transformation should be done with a low concentration of DNA, to minimize uptakeof multiple plasmids into the same cell. The advantage of using only the plasmid originfor retrieval is that any possible background of religated vector is eliminated.

Gap repair is less efficient than targeting to the chromosome. The maximal yield achievedin the authors’ laboratory is ∼1000 recombinants/108 cells. A possible side reactionis joining of the plasmid ends without incorporation of the chromosomal marker. Thisis caused by small (>5 base) repeats near the linear ends (Zhang et al., 2000); it islikely that the Red functions facilitate the short repeat recombination that generates thisbackground. The short repeat recombination can be reduced or eliminated by designingprimers that are free of such repeated sequences.


1.16.13


SUPPORTPROTOCOL 3

SCREENING FOR UNSELECTED RECOMBINANTS

When recombinant frequencies approach 1/1000, direct screening can often be used tofind recombinant colonies from total viable cells plated out nonselectively on LB. Theauthors have successfully used the nonradioactive Roche DIG (digoxigenin) system forcolony hybridization to detect the recombinant bacterial colonies (Thomason, unpub.observ.). For this method to be feasible, the sequence inserted by recombineering mustbe unique to the recombinant and absent in the starting strain. The authors have used a21-nt long DIG-labeled oligonucleotide probe to detect insertion of the same sequence.For larger insertions or fusion proteins such as GFP derivatives, a labeled PCR productor gel-purified fragment could also be used as a probe. Both oligo probes and larger DNAfragments could be radioactively labeled with 32P.

The authors have also successfully isolated unselected recombinants when the geneticchange confers a slow growth phenotype by simply looking for colonies that grow moreslowly than the majority class. Another useful method is the mismatch amplificationmutation assay-PCR (MAMA-PCR; Cha et al., 1992; Swaminathan et al., 2001). MAMA-PCR is capable of identifying single base changes by screening colonies.


Flanking primers for PCR analysis of mutation of interest (also see UNIT 15.1)Additional reagents and equipment for colony hybridization (e.g., UNIT 6.6)

1. Introduce the genetic change of interest (see Basic Protocol 1, steps 1 to 18).

Possible changes include small in-frame deletions or a protein tag. For subtle changes,it is helpful to engineer a restriction-site change that can be detected in a PCR product.For detection by hybridization, confirm beforehand that the sequence does not exist inthe parental bacterial strain—e.g., using a BLAST search (UNIT 19.3; Altschul et al.,1990).

2. Assuming an expected viable cell count in the transformation mix of ∼108, plate thecells nonselectively on rich plates so that the expected number of colonies per plateis ∼500.

Six plates should be adequate if the recombination is efficient. More crowded plates willmean that fewer plates must be screened, but if the plates are too crowded it will be moredifficult to locate positive clones.

3. Screen for recombinants by performing colony hybridization using established proce-dures (e.g., UNIT 6.6). If using a nonradioactive labeling method, follow the conditionssuggested by the manufacturer.

The required length of oligonucleotide probes will depend on the sensitivity of the system.

The authors have detected positives colonies at frequencies as low as 5 × 10−2 to 1 ×10−3.

4. Streak positive candidates to obtain pure clones and retest.

Since the recombination only alters one of several copies of the chromosome existingin a single cell, the colony from that cell will be heterozygous for the allele. Thus, onre-streaking, some fraction of the colonies will not give a positive signal. The signal canagain be detected by colony hybridization. If a new restriction site has been designed intothe construct, perform PCR and cut the product with the appropriate restriction enzymeto detect the recombinant (also see UNIT 3.1).



1.16.14


SUPPORTPROTOCOL 4

SCREENING FOR UNSELECTED PLASMID RECOMBINANTS

With the extremely high oligonucleotide recombination frequencies obtainable in theabsence of mismatch repair (20% to 25% of total viable cells), direct sequencing ofunselected plasmid clones can be used to find recombinants. If the oligonucleotidecarrying the mutation to be introduced creates a C-C mismatch when paired at the targetsite, the recombination can be done in wild-type cells containing the defective prophage,since a C-C mismatch is not repaired. Otherwise, a bacterial strain with prophage andmutant for the mismatch repair system can be used. It is helpful if the mutation tobe inserted creates a restriction site change that can be monitored by digestion of aPCR-amplified fragment covering the region of interest.


Cells expressing Red but mutant for host mismatch repair system oroligonucleotide that creates C-C mismatch when annealed to target DNA strand:e.g., HME63 and/or HME64 (see Table 1.16.1)

High-efficiency cloning strain (lacking the Red system)

1. Perform Basic Protocol 1, introducing both the plasmid and the oligonucleotide byco-electroporation into cells mutant for the host mismatch repair system, or using anoligo that creates a C-C mismatch when annealed to the target DNA strand.

2. After the outgrowth, dilute the electroporation mix into 10 ml broth containing theappropriate antibiotic for plasmid selection and grow the culture overnight.

3. Isolate plasmid DNA from this culture.

4. Using a low DNA concentration, transform the engineered plasmid into a highefficiency cloning strain (lacking the Red system), selecting for drug resistance.Purify colonies.

Use a low DNA concentration to minimize uptake of more than one molecule/cell.

5. Screen to find the mutation, either by direct sequencing or, if there is a restriction sitechange, by amplifying a fragment with PCR and digesting it with the appropriateenzyme(s).

The authors recommend looking at 25 to 50 colonies. This procedure has been successfullyused for plasmid mutagenesis (Thomason, unpub. observ.).

SUPPORTPROTOCOL 5

MODIFYING MULTICOPY PLASMIDS WITH RECOMBINEERING

Multicopy plasmids can be modified by recombineering with efficiencies similar to thoseobtained when targeting the E. coli chromosome. This protocol optimizes the basicprocedure to deal with additional complexities arising when plasmids are targeted (seeCommentary). Addition of both the plasmid and the linear substrate DNA, either double-or single-stranded, by co-electroporation allows better control over the initial ratio ofplasmid molecules to number of cells and minimizes opportunity for plasmid multimerformation. After recombineering, the recombinant species will be contaminated withunmodified parental plasmid and the two species must be separated.


Plasmid to be modified: monomer species, freshly isolated from recA mutant hostsuch as DH5α; determine plasmid DNA concentration by A260

recA mutant bacterial strain expressing Red functions (e.g., DY331 or DY380; seeTable 1.16.1)


1.16.15


Appropriate selective plates, if needed (when selecting for drug resistance, usedrug concentrations appropriate for the multicopy plasmid used)

Additional reagents and equipment for plasmid miniprep (UNIT 1.6) and agarose gelelectrophoresis (UNIT 2.5A)

1. Follow Basic Protocol 1, steps 1 to 13, using a recA mutant strain containing the Redfunctions.

2. In step 14 of Basic Protocol 1, mix the plasmid DNA and the linear DNA, eithersingle-stranded or double-stranded, with the cells. The DNAs will be introducedinto the cells in one electroporation reaction. Include the following electroporationreactions and controls:

a. Induced cells plus plasmid and modifying DNA.

This is the culture that should yield the recombinant plasmids.

b. Induced cells without plasmid or linear DNA.

This is a control to identify contamination in the bacterial culture.

c. Induced cells plus plasmid only.

This control will provide a measure of the plasmid transformation efficiency and serve asa negative control for recombinant selection or screening.

d. Uninduced cells plus plasmid and linear DNA.

This control will confirm that the “recombinants” are dependent on expression of the Redsystem.

If the plasmid is introduced by co-electroporation with the linear substrate, enoughmolecules of plasmid must be added to obtain a high transformation frequency, sincerecombineering will occur only in cells receiving the plasmid. In most cases, ∼10 ngof plasmid DNA per electroporation is sufficient, but it may be necessary to determineempirically the appropriate amount of plasmid DNA to add, since the transformationefficiency of the plasmid being used may differ. Ideally, each cell should receive aboutone plasmid molecule.

If the plasmid is already resident in the cell, add only the modifying linear DNA at thisstep.

3. Perform electroporation as in step 15 of Basic Protocol 1. After electroporation,follow step 16 for outgrowth, using the longer 2 hr time.

4. Add 9 ml LB medium and the appropriate amount of antibiotic for plasmid selection;let the culture grow overnight at 30◦ to 32◦C.

5. The following day, isolate plasmid DNA from the culture with a standard miniprepprocedure (UNIT 1.6).

If the recombinant has been designed to remove a unique restriction site, digest severalmicroliters of the DNA with this enzyme. This will help to eliminate the inevitable back-ground of unmodified parental plasmid, thus enriching for the recombinant population.

6. Transform the plasmid miniprep DNA or the clean restriction digest into a standardrecA cloning strain such as DH5α at a low DNA concentration (less than one DNAmolecule/cell).

Electroporation is highly recommended, since it is much more efficient than chemicaltransformation.

7. After a 2-hour outgrowth, plate for single colonies on the appropriate media. Plateselectively to select recombinant colonies, or nonselectively on LB plates (plus



1.16.16


antibiotic for plasmid selection) and screen colonies for the desired phenotype.Isolate the recombinant plasmid DNA from about ten candidate colonies and screenfor a monomer species by visualization with agarose gel electrophoresis (UNIT 2.5A).

Possible methods of screening include restriction digestion, identification of plasmidswith altered size as assayed by migration on agarose gels, sequencing, and PCR analysis(i.e., MAMA-PCR; Cha et al., 1992).

8. If a multimeric plasmid that contains the desired modification has been identified,convert it to a monomer species by first digesting it with a unique restriction enzymeand subsequently ligating under conditions favoring an intramolecular reaction (i.e.,a low DNA concentration). Verify the presence of the desired modification on themonomer plasmid.

BASICPROTOCOL 2

REMOVAL OF THE PROPHAGE BY RECOMBINEERING

Once the mutational changes are introduced, the defective λ prophage can be removed ifnecessary. This is done by another Red-mediated recombination reaction. Alternatively,the prophage can be removed by P1 transduction. In both cases, the desired recombinantcan be selected on minimal plates since it will grow in the absence of biotin.

Materials

Oligonucleotide primers for amplifying the bacterial attB site:5′ GAGGTACCAGGCGCGGTTTGATC 3′5′ CTCCGGTCTTAATCGACAGCAAC 3′

E. coli K12 strain lacking the prophage (e.g., W3110), but containing the attB andbiotin (bio) genes

M63 minimal glucose plates (see recipe) with and without biotin

Additional reagents and equipment for recombineering (see Basic Protocol 1)

1. Amplify the bacterial attB site by PCR using 50 pmol of each of the primers listedabove, with an E. coli K12 strain lacking the prophage (e.g., W3110) as template(the PCR product is ∼2.5 kb), and the following program:

1 cycle: 2 min 94◦C (denaturation)9 cycles: 15 sec 94◦C (denaturation)

30 sec 65◦C (annealing)3 min 68◦C (extension)

19 cycles: 15 sec 94◦C (denaturation)30 sec 65◦C (annealing)3 min(adding 5 sec/cycle) 68◦C (extension)

1 cycle: 7 min 68◦C (extension)1 cycle: indefinite 4◦C (hold)

The technique used here is “colony PCR” in which the PCR reaction is prepared withoutallowing any volume for the template DNA; a fresh colony of the E. coli K12 strain isthen touched with a sterile inoculating loop and mixed into the PCR reaction.

2. Delete the prophage by recombineering (see Basic Protocol 1, steps 1 to 18) usingthe attB PCR product for recombination.

3. Wash the cells in minimal salts twice and resuspend them in the same medium forplating. Select for the desired recombinant on minimal glucose plates lacking biotin


1.16.17


but containing vitamin B1 by incubating 2 hr at 32◦C then shifting to 42◦C untilcolonies appear.

LB has trace amounts of biotin, enough to complement a bio mutant for growth; thereforethe cells must be washed free of LB for this selection to be successful. Those cells in whichthe prophage has been deleted will be bio+, since the PCR product brings in a wild-type(wt) biotin gene, and will grow at 42◦C, since the removal of the prophage makes thecells temperature resistant. As a control, confirm that the prophage-containing strain,subjected to the same washes, does not plate on minimal glucose plates lacking biotin,but does plate when biotin is present.

The 32◦C incubation allows time for the recombinant chromosomes to segregate awayfrom those still containing the prophage, which expresses a killing function at hightemperature (see Commentary). Streak the recombinant colonies to purify them. As anadditional confirmation that the prophage has been deleted, the same PCR primers canbe used to confirm the presence of the 2.5-kb band in the purified strain.

ALTERNATEPROTOCOL 1

RECOMBINEERING WITH AN INTACT λ PROPHAGE

Basic Protocol 1 describes a method for recombineering using a defective lambdoidprophage. It is also possible to recombineer using an intact λ prophage (Court et al.,2003). If the phage of interest has a temperature-sensitive cI857 repressor, intact exo,beta, and gam genes, and is able to lysogenize the host, it can exist in the prophage stateand can be induced by high temperature to express the recombination functions. Sucha phage will provide the necessary functions for recombineering; it can also itself serveas a target for such engineering. Since the recombineering efficiency is lower with thismethod than with the defective prophage, a selection for the recombinants should beapplied. To recombineer using an intact phage, construct and confirm a bacterial lysogen(Arber et al., 1983) with the phage of choice. Once the phage exists in the prophage state,it can be induced by a temperature shift, as described below, and the PCR product oroligo containing the desired genetic change can be introduced by electroporation.


Bacterial lysogen carrying the λ cI857 bacteriophage of choice as a prophageChloroform1× TM buffer (APPENDIX 2)

82-mm nitrocellulose filters, sterile39◦C water bath

Additional reagents and equipment for plating λ phage to generate plaques (plaquepurification; UNIT 1.11)

1. Grow the host bacterial strain (lysogen) to mid-log phase at 32◦C (see Basic Proto-col 1, step 6).

2. Induce recombination functions, electroporate cells, and plate to determine viablecell counts (see Basic Protocol 1, steps 7 to 18, but reduce the induction time to 4 to5 min in step 7).

The induction time must be shorter to prevent lytic phage replication and resultant cellkilling. The shorter induction time means that lower levels of the recombination functionsare produced. The only situation in which the 15-min induction time should be used iswhen changes are being targeted to the bacteriophage chromosome itself (steps 3b and4a).

If the mutation of interest is targeted to the bacterial chromosome

3a. Select for recombinants by plating an entire electroporation mix on one selective plate(because recombinant levels are reduced by the lower induction time). Use a sterile



1.16.18


82-mm diameter nitrocellulose filter atop a rich (LB) plate and incubate >3 hr at 30◦to 32◦C, then transfer the filter to the appropriate drug plate using sterile forceps.

The number of recombinants is generally less than 500 per electroporation mix. Usually,approximately half of the surviving cells will have spontaneously lost the prophage. It ispossible to screen for non-lysogens by testing candidate colonies for their ability to plateλ, since the prophage renders the cells immune to phage infection; the cured cells willalso be viable at 42◦C while those containing the prophage will not (see Commentary).

4a. Use PCR (UNIT 15.1) and subsequent DNA sequencing (Chapter 7) to confirm themutation.

If mutations have been targeted to the bacteriophage itself

In this case, the 15-min induction time can be used.

3b. Dilute the electroporation mix into 5 ml LB medium and aerate by shaking in ashaking water bath 90 min at 39◦C. Add 0.25 ml chloroform to complete cell lysisand release the phage particles. Dilute the lysate and plate for single plaques (seeUNIT 1.1).

4b. Plaque-purify the positive candidates (UNIT 1.11). Resuspend a plaque in 50 µl ofsterile water and use 20 µl of this suspension as template for a PCR reaction (UNIT

15.1; reduce the amount of water accordingly). Resuspend another plaque from thesame plate in 1 ml of 1× TM buffer to grow a stock. Reconfirm the mutation aftergrowing the stock.

ALTERNATEPROTOCOL 2

TARGETING AN INFECTING PHAGE λ WITH THE DEFECTIVEPROPHAGE STRAINS

Sometimes it may be useful to target genetic changes to bacteriophage λ derivatives.A strain carrying the Red system on the prophage or a plasmid can be infected with aphage and recombineering can then be targeted to the incoming phage chromosome. Aprocedure for this method follows. Ideally the construction should be designed so thatthe plaque morphology of the recombinant phage will differ from that of the parent. Forexample, a PCR product able to both introduce the mutation of interest and correct aknown mutation (such as an amber or temperature-sensitive allele) can be recombinedonto a phage containing the known mutation. Select for correction of the known mutationand screen among these recombinants for the mutation of interest. If no selection exists,plaque hybridization can sometimes be used to identify recombinant phages. While theauthors have only tested this method for phage λ, theoretically it may be possible tointroduce genetic changes onto the chromosome of any phage able to propagate in thedefective prophage host (Oppenheim et al., 2004).


10% (w/v) maltose stock solution, filter sterilized1× TM buffer (APPENDIX 2)High-titer lysate of the bacteriophage to be engineeredPCR product with desired sequence changes and flanking homology to the target

on the phage chromosomeLambda plates and lambda top agar (see UNIT 1.1, adjust NaCl to 5 g per liter in both

the plates and the top agar)ChloroformAppropriate bacterial indicator strain

Additional reagents and equipment for working with λ bacteriophages(UNITS 1.9-1.13)


1.16.19


1. Grow host strain with defective prophage to mid-log phase at 32◦C (see BasicProtocol 1, step 6), except supplement the LB medium with 0.4% maltose.

Maltose induces the phage receptor on the bacterial cell surface, ensuring efficientadsorption of the phage to the host cells.

2. Harvest the cells by centrifuging 7 min at 4600 × g, 4◦C. Resuspend pellet in 1 mlof 1× TM buffer. Infect the cells with the phage to be engineered at a multiplicity of1 to 3 phages per cell.

The cells will be at ∼1 × 108/ml before concentration. UNITS 1.9-1.13 contain protocolsfor working with λ bacteriophages.

3. Let the phage adsorb to the cells for 15 min at room temperature.

Phages other than λ may require different adsorption conditions.

4. Transfer the infected cells to 5 ml of 42◦C LB medium and shake vigorously for 15min. At end of incubation, chill rapidly on ice.

This serves to both induce the Red functions and allow phage infection to proceed.

5. Make electrocompetent cells and introduce transforming DNA (i.e., introduce PCRproduct with mutation of interest; see Basic Protocol 1, steps 8 to 14).

6. Dilute electroporation mix into 5 ml warm LB medium and shake vigorously for 90min at 42◦C to allow the phage to complete a lytic cycle. Add 0.25 ml chloroformto completely lyse infected cells. Plate phage on lambda plates with lambda topagar (see UNIT 1.11) using the appropriate bacterial indicator strain as a host. Applya selection, if possible, or plate non-selectively and screen for the desired mutationwith plaque hybridization.

Amber mutations can be specifically selected (Oppenheim et al., 2004).

REAGENTS AND SOLUTIONSUse Milli-Q purified water or equivalent in all recipes and protocol steps. For common stocksolutions, see APPENDIX 2; for suppliers, see APPENDIX 4.

M63 minimal glucose plates

Per liter:3 g KH2PO4

7 g K2HPO4

2 g (NH4)SO4

0.5 ml of 1 mg/ml FeSO4

0.2% (v/v) glucose0.001% (w/v) biotin (omit for control)1 ml 1% (w/v) vitamin B1 (thiamine)1 ml 1 M MgSO4

15 g agarPour 40 ml of the agar-containing medium per plate.Store up to several months at 4◦C.

M63 minimal glycerol plates with sucrose

Per liter:3 g KH2PO4

7 g K2HPO4

2 g (NH4)SO4

0.5 ml of 1 mg/ml FeSO4

continued



1.16.20


0.2% (v/v) glycerol5% (w/v) sucrose0.001% (w/v) biotin1 ml 1% (w/v) vitamin B1 (thiamine)1 ml 1 M MgSO4

15 g agarPour 40 ml of the agar-containing medium per plate.Store up to several months at 4◦C.

COMMENTARY

Background InformationBacteriophage λ encodes three genes im-

portant for recombineering. The exo and betgenes, respectively, encode a 5′ to 3′ double-strand exonuclease, Exo, and a single strandannealing protein, Beta, which together can re-combine a double-stranded PCR product withshort flanking homologies into the desiredgenetic target. The λ gam gene encodes a pro-tein, Gam, that inhibits the RecBCD enzyme,which will otherwise degrade linear DNA in-troduced into the bacterial cell. Only the Betasingle-strand annealing function is requiredto recombine single-stranded oligos contain-ing the desired alterations. A cryptic lambdoidprophage, rac, is present in some strains ofE. coli, and encodes RecE and RecT functionsthat are analogous to Exo and Beta, respec-tively. Unlike some other in vivo geneticengineering methods (Russell et al., 1989), re-combineering does not require the host recAfunction. A strain mutant for recA providesmore controllable recombination, since thestrain is recombination proficient only whenthe phage functions are induced. A more de-tailed discussion of the molecular mechanismof recombineering can be found in Court et al.(2002).

Recently the Red genes have been movedto several plasmids (Table 1.16.3; Datta et al.,2006) having different DNA replication ori-gins. Here, the essential control elements ofthe prophage system are retained and recom-bination functions are induced by a temper-

Table 1.16.3 Plasmids Containing the Red System Under cI857 Control

Plasmid designation Plasmid originApproximate copy

number/cell

pSIM5(CmR)pSIM6(AmpR)

pSC101ts 16

pSIM7(CmR)pSIM8(AmpR)

pBBR1 30-40

pSIM9(CmR) pRK2ts 20-40

ature shift as in Basic Protocol 1. The plas-mids are especially useful when one wantsto create a mutation in a particular bacterialstrain, rather than create the mutations in theprophage-containing strains and subsequentlymove them into a different background.


Induction timesWhen inducing the recombination func-

tions from the defective prophage, the properinduction time is essential. Longer inductiontimes will cause decreased cell viability, sincethe prophage Kil function is also induced bythe temperature shift. The Gam function, nec-essary for efficient transformation of dsDNA,is also toxic to the cells (Sergueev et al., 2001).

Storage of induced cellsIt is also important to use the induced cells

promptly, since the induced phage functionswill decay over time, especially at 32◦C. Al-though one pioneering laboratory member re-ports that induced cells can be successfullystored on ice for several hours before electro-poration, the authors do not recommend thisprocedure. As detailed in Basic Protocol 1,competent cells may be frozen in 15% glyc-erol, although their recombineering efficiencyhas been less than that of the freshly preparedcells.


1.16.21


Amplification of drug cassettesIt is strongly recommended that each drug

cassette be amplified with only one standardset of primers. Confusion has arisen in the au-thors’ laboratory when individuals have de-signed their own primers for the drug cassettes,because a strain containing a drug cassette in-serted by recombineering may be used lateras a PCR template for that drug marker. Us-ing a standard set of primers ensures that thehomology used to amplify the drug cassetteis always present, whatever strain is used forPCR amplification. Remember that it is best tohave a promoter, the open reading frame, anda transcriptional terminator on each cassette.Standard priming sequences for the commonlyused cassettes are listed in Table 1.16.2.

The cat-sacB elementThe cat-sacB DNA segment described in

Support Protocol 1 is amplified from plasmidpEL04, previously known as both pKO4 andpcat-sacB, or from the strain DY441, usingcolony PCR. If a different antibiotic resistancegene is required, the cat gene on pcat-sacBcan be replaced with another drug marker. Use50 bases of homology upstream of the 5′ catprimer and downstream of the 3′ cat primerto cross in the other marker. The sequence ofpEL04 is available from the Court Laboratory([email protected]). Since the cat-sacB PCRproduct is 3264 bp, it is not always easy to am-plify. Optimized PCR conditions are detailedabove, but other conditions and any other high-fidelity enzyme should also work. This is reallyan empirical problem. The biggest factors, ifsufficient extension times and appropriate tem-peratures are used, are the PCR machine itselfand the polymerase. Adjusting the Mg2+ con-centration may also improve yield. There hasbeen some confusion regarding the primers foramplification of the cat-sacB cassette. An op-timal primer pair has been designed for ampli-fication of this DNA, and it is strongly recom-mended that only this set of primers be used.Please note that both primer sequences are dif-ferent from those previously given in this pro-tocol.

Detecting recombinantsIf one has engineered a drug marker into

the DNA, drug resistance will serve as the se-lection. The two-step cat-sacB replacement al-lows precise modification without leaving anantibiotic-resistance cassette or other markerbehind. Using colony hybridization with a la-beled oligo probe has also been successful,although a unique sequence hybridizing only

to the probe must be inserted (or a unique se-quence must be created by a deletion) for thismethod to be an option.

Mismatch repair minus conditions foroligonucleotide recombination

It has been reported (Costantino and Court,2003) that extremely high levels of recom-binants can be obtained when recombineer-ing with oligos if the bacterial strain is mu-tant for the host methyl-directed mismatchrepair system (the mut HLS system). Underthese conditions ∼20% to 25% of the to-tal viable cells are recombinant. Strains withthe prophage and mutations in the mismatchrepair system are available from the Court lab-oratory: these are HME63 and HME64 andcontain a mutS<>amp and an uvrD<>kanmutation, respectively. Either mutation willeliminate mismatch repair; the latter strain canbe used for engineering onto plasmids express-ing ampicillin resistance. The same elevatedlevel of recombination is obtained in mismatchrepair proficient strains if the incoming oligocreates a C-C mismatch when annealed to thetarget DNA, since C-C mispairs are not subjectto mismatch repair.

Counter-selectionsThe cat-sacB cassette is a counter-

selection; it can be selected either for (by chlo-ramphenicol resistance) or against (sucrosesensitivity). Several such counter-selectionsare available and may prove useful for par-ticular situations; tetracycline resistance is an-other example (Bochner et al., 1980; Maloyand Nunn, 1981). If removing a bacteriostaticdrug marker (such as tetracycline), enrichmentfor recombinants is possible using ampicillin.Nonrecombinant bacteria will still express re-sistance to the tet marker encoded on the chro-mosome; when the electroporated culture ispropagated in the presence of both tetracy-cline and ampicillin, only nonrecombinantscells will grow; these nonrecombinants will bekilled by the ampicillin (Murphy et al., 2000).Recombinant cells will not grow but will not bekilled. 2-Deoxy-D-galactose is a toxic analogof galactose on which only mutants of galac-tokinase, the product of the galK gene, willgrow (Alper and Ames, 1975). The galK se-lection has recently been adapted for use withBACs (Warming et al., 2005). Other counter-selections may be devised.

In vivo assembly with overlapping oligosComplementary oligos will anneal in vivo

when introduced by electroporation into cells



1.16.22


expressing Exo and Beta protein (Yu et al.,2003). Multiple overlapping oligos can be usedto build moderately sized DNA products (100to 150 bp) in an in vivo reaction similar to PCRassembly (Stemmer et al., 1995). By addingflanking homologies to chromosomal targets,these overlapping oligos are restored to lineardsDNA in vivo and recombined into the target.

Anticipated ResultsAs discussed above, ideally, transforma-

tion with dsDNA and ssDNA can approachfrequencies of 0.1% and 25%, respectively.Because of this high efficiency, there shouldrarely if ever be experiments where no recom-binants are found. However, in practice, re-combineering frequency may be partially con-text dependent; certain areas of the chromo-some appear “hotter” for recombination thanothers (Ellis et al., 2001). If recombinants arenot obtained, check the construct design andredesign primers if necessary, as this is onereason for failure. If the recombineering reac-tion does not work, a control experiment usingknown strains and oligos or PCR products, asdescribed in Yu et al. (2000), Ellis et al. (2001),and Costantino and Court (2003), is recom-mended to verify that the predicted numberof recombinants are obtained in these controlreactions. Most recombineering failures occurbecause the protocol is not executed carefullyor properly.

Recombineering onto plasmidsWhen considering using recombineering to

modify a plasmid, it is important to first de-termine whether it is the appropriate methodto use. Recombineering targeted to a plasmidworks as efficiently as when it is used to targetthe bacterial chromosome, and the same guide-lines apply. This means that when a lagging-strand oligo is used for recombineering in theabsence of mismatch repair, point mutationsand modifications of a few bases can be madeat a frequency that allows their isolation inthe absence of selection. Mismatch repair canbe eliminated either by use of the mismatch re-pair mutant strains or by creation of a C-C mis-pair when the oligo is annealed to the target.For insertion and deletion of larger segmentsof DNA, however, standard cloning techniquesmay be preferable, since in vitro cloning meth-ods are more efficient than recombineering forthese reactions. Recombineering-mediated in-sertion and deletion of larger pieces of DNAon plasmids occurs at a lower efficiency thancreation of point mutations; thus a selectionor way to separate recombinant plasmids from

unmodified parental plasmids is required. Ifthe goal is simply to insert or remove a seg-ment of DNA and the exact junctions are notcritical, in vitro cloning will often serve thepurpose well. Recombineering, however, al-lows creation of a precise nucleotide sequenceat the junction between two DNAs, which isnot always possible with standard cloning.

The recombinant species can often beenriched for by destruction of the parentalplasmid molecule. However, the formationof circular multimers during recombineeringcomplicates isolation and analysis of recom-binants, since only one of the target sites on amultimeric plasmid may be modified. Becauseof the propensity of recombinant plasmids tohave multimerized, destroying parental plas-mid will also result in loss of recombinant plas-mids that are not modified at all target sites. Itmay be preferable to convert the plasmid pop-ulation to monomer first by digesting it with arestriction enzyme that cuts at a unique site inboth parental and recombinant plasmids, thenligating under dilute conditions. The result-ing monomer population can then be enrichedfor recombinants by digestion of the monomerparental molecules. It should be noted that thecircular multimer plasmid species arising dur-ing recombineering are not identical to the lin-ear multimers observed by Cohen and Clark(1986) after expression of the λ Gam func-tion. Plasmid multimers that form during re-combineering are circular and are found in therecombinant plasmid population after oligo re-combination when only the λ Beta protein hasbeen used to catalyze the reaction (Thomasonet al., 2007).

The plasmid DNA can be introduced intothe cell at the same time as the linear substrateDNA (co-electroporation), or it can be resi-dent in the cell prior to electroporation. Eachoption has advantages and disadvantages. Co-electroporation allows more control over thenumber of plasmids introduced per cell, but isless convenient if the plasmid is large (>10 kb).In the protocols described here, directions aregiven for co-electroporation. To target a resi-dent plasmid, the episome should have beenintroduced into a recombineering-proficientrecA mutant host strain.

Engineering onto BACsRed recombineering has also been opti-

mized for engineering bacterial artificial chro-mosomes (Copeland et al., 2001; Lee et al.,2001; Swaminathan and Sharan, 2004), whichare able to accommodate hundreds of kilo-bases of foreign DNA. DY380 is the bacterial


1.16.23


host of choice for use with BACs; it is aDH10B derivative containing the defectiveprophage. The recombineering plasmids listedin Table 1.16.3 may also be useful in manipu-lating BACs.

Mutating essential genesMutation of an essential gene usually re-

sults in cell death. Such a mutation can berecovered in a diploid state; however, the cellwill maintain a wild-type copy of the gene aswell as a mutant version. This diploid state canusually be detected by PCR analysis.

Moving the prophage to a differentbackground

If a different host is required for recombi-neering, the defective prophage can be movedby P1 transduction (Miller, 1972; Yu et al.,2000). Grow a P1 lysate on DY329 and trans-duce the strain of choice. Select for tetracy-cline resistance and screen for temperaturesensitivity or an inability to grow in the ab-sence of biotin. Check the genotype of thestrain first and supplement the minimal platesto complement any additional auxotrophieswhen performing the biotin screen.

Time ConsiderationsThe basic recombineering protocol can be

executed in one day, with bacterial culturesstarted the evening before. The recombinantsmay take several days to grow on the selectiveplates, and it may take another day or so toconfirm the recombinants.

Literature CitedAlper, M.D. and Ames, B.N. 1975. Positive selec-

tion of mutants with deletions of the gal-chl re-gion of the Salmonella chromosome as a screen-ing procedure for mutagens that cause deletions.J. Bacteriol. 121:259-266.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W.,and Lipman, D.J. 1990. Basic local alignmentsearch tool. J. Mol. Biol. 215:403-410.

Arber, W., Enquist, L., Hohn, B., Murray, N.E.,and Murray, K. 1983. Experimental methods foruse with lambda. In Lambda II (R. Hendrix, J.Roberts, F. Stahl, and R. Weisberg, eds.) pp.433-471. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

Blomfield, I.C., Vaughn, V., Rest, R.F., andEisenstein, B.I. 1991. Allelic exchange inEscherichia coli using the Bacillus subtilis sacBgene and a temperature-sensitive pSC101 repli-con. Mol. Microbiol. 5:1447-1457.

Bochner, B.R., Huang, H.C., Schieven, G.L., andAmes, B.N. 1980. Positive selection for lossof tetracycline resistance. J. Bacteriol. 143:926-933.

Cha, R.S., Zarbl, H., Keohavong, P., and Thilly,W.G. 1992. Mismatch amplification mutationassay (MAMA): Application to the c-H-rasgene. PCR Methods Appl. 2:14-20.

Cohen, A. and Clark, A. J. 1986. Synthesis of linearplasmid multimers in Escherichia coli K-12. J.Bacteriol. 167:327-35.

Copeland, N.G., Jenkins, N.A., and Court, D.L.2001. Recombineering: A powerful new toolfor mouse functional genomics. Nat. Rev. Genet.2:769-779.

Costantino, N. and Court, D.L. 2003. Enhancedlevels of lambda Red-mediated recombinants inmismatch repair mutants. Proc. Natl. Acad. Sci.U.S.A. 100:15748-15753.

Court, D.L., Sawitzke, J.A., and Thomason, L.C.2002. Genetic engineering using homologousrecombination. Annu. Rev. Genet. 36:361-388.

Court, D.L., Swaminathan, S., Yu, D., Wilson, H.,Baker, T., Bubunenko, M., Sawitzke, J., andSharan, S.K. 2003. Mini-lambda: A tractablesystem for chromosome and BAC engineering.Gene 315:63-69.

Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes inEscherichia coli K-12 using PCR products.Proc. Natl. Acad. Sci. U.S.A. 97:6640-6645.

Datta, S., Costantino, N., and Court, D.L. 2006.A set of recombineering plasmids for gram-negative bacteria. Gene 379:109-115.

Ellis, H.M., Yu, D., DiTizio, T., and Court, D.L.2001. High efficiency mutagenesis, repair, andengineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci.U.S.A. 98:6742-6746.

Gay, P., Le Coq, D., Steinmetz, M., Berkelman, T.,and Kado, C.I. 1985. Positive selection proce-dure for entrapment of insertion sequence ele-ments in Gram-negative bacteria. J. Bacteriol.164:918-921.

Lee, E.C., Yu, D., Martinez de Velasco, J.,Tessarollo, L., Swing, D.A., Court, D.L.,Jenkins, N.A., and Copeland, N.G. 2001. Ahighly efficient Escherichia coli–based chro-mosome engineering system adapted for re-combinogenic targeting and subcloning of BACDNA. Genomics 73:56-65.

Maloy, S.R. and Nunn, W.D. 1981. Selection forloss of tetracycline resistance by Escherichiacoli. J. Bacteriol. 145:1110-1111.

Miller, J.M. 1972. Generalized transduction: Useof P1 in strain construction. In Experimentsin Molecular Genetics (J.M. Miller, ed.) pp.201- 205. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

Murphy, K.C., Campellone, K.G., and Poteete,A.R. 2000. PCR-mediated gene replacement inEscherichia coli. Gene 246:321-330.

Oppenheim, A.B., Rattray, A.J., Bubunenko, M.,Thomason, L.C., and Court, D.L. 2004. In vivorecombineering of bacteriophage λ by PCR



1.16.24


fragments and single-strand oligonucleotides.Virology 319:185-189.

Russell, C.B., Thaler, D.S., and Dahlquist, F.W.1989. Chromosomal transformation of Es-cherichia coli recD strains with linearized plas-mids. J. Bacteriol. 171:2609-2613.

Sergueev, K., Yu, D., Austin, S., and Court, D. 2001.Cell toxicity caused by products of the PL operonof bacteriophage lambda. Gene 272:227-235.

Stemmer, W.P., Crameri, A., Ha, K.D., Brennan,T.M., and Heyneker, H.L. 1995. Single-step as-sembly of a gene and entire plasmid from largenumbers of oligodeoxyribonucleotides. Gene164:49-53.

Swaminathan, S. and Sharan, S.K. 2004. Bacte-rial artificial chromosome engineering. MethodsMol. Biol. 256:89-106.

Swaminathan, S., Ellis, H.M., Waters, L.S., Yu, D.,Lee, E-C., Court, D.L., and Sharan, S.K. 2001.Rapid engineering of bacterial artificial chromo-somes using oligonucleotides. Genesis 29:14-21.

Thomason, L.C., Myers, R.S., Oppenheim, A.,Costantino, N., Sawitzke, J.A., Datta, S.,Bubunenko, M., and Court, D.L. 2005. Re-combineering in prokaryotes. In Phages: TheirRole in Bacterial Pathogenisis and Biotechnol-ogy (M.K. Waldor, D.I. Friedman, and S.L.Adhya, eds.) pp. 383-399. ASM Press,Herndon, Va.

Thomason, L.C., Costantino, N., Shaw, D.V., andCourt, D.L. 2007. Multicopy plasmid modifica-tion with phage λ Red recombineering. Plasmid.In press.

Yu, D., Ellis, H.M., Lee, E.C., Jenkins, N.A.,Copeland, N.G., and Court, D.L. 2000. An effi-cient recombination system for chromosome en-gineering in Escherichia coli. Proc. Natl. Acad.Sci. U.S.A. 97:5978-5983.

Yu, D., Sawitzke, J.A., Ellis, H., and Court, D.L.2003. Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a re-combination intermediate. Proc. Natl. Acad. Sci.U.S.A. 100:7207-7212.

Warming, S., Costantino, N., Court, D.L., Jenkins,N.A., and Copeland, N.G. 2005. Simple andhighly efficient BAC recombineering using galKselection. Nucl. Acids Res. 33:e36.

Zhang, Y., Muyrers, J.P., Testa, G., and Stewart,A.F. 2000. DNA cloning by homologous recom-bination in Escherichia coli. Nature Biotechnol.12:1314-1317.

Contributed by Lynn Thomason, DonaldL. Court, Mikail Bubunenko, NinaCostantino, Helen Wilson, SimantiDatta, and Amos Oppenheim

National Cancer Institute at FrederickFrederick, Maryland

UNIT 1.17E. coli Genome Manipulation by P1Transduction

Lynn C. Thomason,1 Nina Costantino,1 and Donald L. Court1

1National Cancer Institute at Frederick, Frederick, Maryland

ABSTRACT

This unit describes the procedure used to move portions of the E. coli genome fromone genetic variant to another. Fragments of ∼100 kb can be transferred by the P1bacteriophage. The phage is first grown on a strain containing the elements to be moved,and the resulting phage lysate is used to infect a second recipient strain. The lysate willcontain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed byenzymes of the recipient strain, will incorporate the bacterial fragments into the recipientchromosome. Curr. Protoc. Mol. Biol. 79:1.17.1-1.17.8. C© 2007 by John Wiley & Sons,Inc.

Keywords: transduction � E. coli bacteriophage P1 � genetic recombination

INTRODUCTION

P1 transduction is a useful genetic procedure for moving selectable mutations of interestfrom one E. coli strain to another. P1 is a bacteriophage with a rather sloppy packagingmechanism, and during growth and encapsidation of its DNA into the phage head, it oc-casionally packages the DNA of its bacterial host rather than its own phage chromosomeinto the protein capsid. This property of the P1 phage means that when a P1 lysate is made,the individual particles in the lysate contain either packaged phage DNA or packagedbacterial DNA. When this lysate is used to infect a second (recipient) host, DNA piecesfrom the original host chromosome are transferred into the new strain, where they canrecombine and be permanently incorporated into the chromosome of the second strain,an event called transduction. Successful P1 transduction of any one marker from a uniquelocation in one bacterial strain to a second strain is a relatively rare event, so selectionis required. P1 can carry two or more different genetic markers in the same transducingparticle DNA, and at some frequency both of these markers can be recombined into therecipient strain. Such markers are said to be linked by P1 transduction, i.e., they are closeenough to be co-transduced. Proximity of the selectable marker to the allele of interest isimportant, because nearer alleles will co-transduce more frequently. The allele of interestmight be a (defective) gene with an antibiotic marker cassette insertion, or it might be agene with a point mutation that is closely linked to a selectable antibiotic marker.

To execute the Basic Protocol, the P1 lysate is grown on a host containing a genetic variantof interest and this lysate is then used to infect a second strain. For the nonexpert, it isbest to use a virulent phage mutant, P1vir, to produce the lysate because the vir mutationprevents the phage from generating P1 lysogens among transductants. The Basic Protocoldescribes growth of a liquid lysate, which is easier to carry out than a plate lysate andgenerally gives good results. The Support Protocol describes the procedure used totiter the P1 stock, which is usually done only if the transduction is not successful. TheAlternate Protocol provides a method for preparing a plate lysate, which may be usedif the starting P1 preparation has a low titer. Plate lysates often yield higher-titer stocksthan liquid lysates.

Current Protocols in Molecular Biology 1.17.1-1.17.8, July 2007Published online July 2007 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142727.mb0117s79Copyright C© 2007 John Wiley & Sons, Inc.


1.17.1

Supplement 79

E. coli GenomeManipulation byP1 Transduction

1.17.2


BASICPROTOCOL

PREPARATION OF P1 LIQUID LYSATE AND P1 TRANSDUCTION

This protocol describes the two-part procedure: preparation of a liquid P1 lysate, followedby transduction. Each procedure requires a freshly grown bacterial overnight culture.Once the cultures are prepared, the procedures are simple and each requires only a fewhours, so that both can be performed in the same day.

Materials

LB broth (UNIT 1.1)E. coli nonlysogenic donor strain (containing the genetic marker to be transduced)20% glucose stock solution1 M CaCl2109 to 1010 pfu/ml P1 stock, a virulent P1 mutant preferred (e.g., P1vir)Chloroform (CHCl3)E. coli recipient strain (to be transduced)Tris-magnesium (TM) buffer: 10 mM MgSO4/10 mM Tris·Cl, pH 7.4 (APPENDIX 2)P1 salts solution: 10 mM CaCl2/5 mM MgSO4

1 M sodium citrateSelective plates (see UNIT 1.1) with 5 mM sodium citrate (recommended); e.g.,

minimal plates if selecting for prototrophy or rich LB plates containing theappropriate antibiotic (depending on drug cassette used):30 µg/ml ampicillin30 µg/ml kanamycin10 µg/ml chloramphenicol12.5 µg/ml tetracycline (6.25 µg/ml if sodium citrate is added)50 µg/ml spectinomycin

50-ml Erlenmeyer flasks, preferably baffled18 × 150–mm glass culture tubesShaking water bath or test-tube roller at appropriate temperature (usually 32◦ to

37◦C)35-ml plastic centrifuge tubesRefrigerated low-speed centrifuge with Sorvall SA-600 rotor (or equivalent)5-ml screw-cap vials

NOTE: All material coming in contact with E. coli must be sterile.

Prepare liquid P1 lysate1. Inoculate 5 ml LB broth with the E. coli donor strain (from which the P1 lysate is to

be made). Incubate overnight at the appropriate temperature.

Throughout this protocol, the optimal temperature for cell growth is 37◦C, unless atemperature-sensitive strain is used. Temperature-sensitive strains should be cultured at30◦ to 32◦C.

To perform transduction on the same day, grow the recipient strain (step 9) at the sametime.

2. Dilute the overnight donor culture 1:100 into 5 ml LB broth containing 50 µl 20%glucose (final concentration 0.2%) and 25 µl of 1 M CaCl2 (final concentration 5mM) in a 50-ml Erlenmeyer flask or 18 × 150–mm glass culture tube.

3. Incubate 30 to 45 min at 30◦ to 37◦C with shaking.

Use either a 50-ml Erlenmeyer flask in a shaking water bath or a standard culture tubewith adequate aeration in a test-tube roller. If the strain is temperature sensitive, use thelower temperature and the longer time.


1.17.3


4. Add 100 µl of a recently prepared 109 to 1010 pfu/ml P1vir stock.

A good titer for a P1 stock is 109 to 1010 pfu/ml. If the stock has recently been usedsuccessfully and does not contain CHCl3 (which reduces the viability of the stock; seestep 6), it is probably unnecessary to determine the titer.

5. Continue to incubate, with shaking, until the culture lyses (∼3 hr).

The culture may not lyse and clear completely, but clumps of floating debris should bevisible when it is held up to the light. An unlysed culture looks smooth and silky whenswirled; a lysed culture does not.

6. Add a few drops of CHCl3 and continue shaking a few minutes more.

The CHCl3 ensures complete cell lysis and kills the bacteria. CHCl3 does not harm theP1 phage at this step, but care should be taken to avoid transferring the CHCl3 to thestorage tube during step 8, because storing the phage in the presence of CHCl3 can resultin decreased viability of the stock over time.

7. Transfer the supernatant to a 35-ml centrifuge tube and centrifuge 10 min at ∼9200× g, 4◦C.

8. Pour off the supernatant (the P1 lysate) into a screw-cap 5-ml tube. Label it with thestrain on which the lysate was grown (e.g., P1vir·W3110) and the date, and store upto several years at 4◦C.

The lysate can be further purified by passage through a sterile 0.45-µm filter syringe aftercentrifugation. The filter will remove any residual bacteria.

The lysate can be stored for several years at 4◦C and still retain adequate titer. If in doubt,either spot titer as described in the Support Protocol or make a new lysate.

Perform P1 transduction9. Inoculate 5 ml LB broth with the E. coli recipient strain. Incubate overnight at the

appropriate temperature.

If the strain does not have temperature-sensitive elements, use 37◦C.

10. The following day, centrifuge 1.5 ml of the cell culture in a microcentrifuge tube 2min at maximum speed, room temperature, to pellet the cells.

11. Discard the medium and resuspend the cells at one-half the original culture volumein sterile P1 salts solution.

This gives 0.75 ml suspended cells, which is adequate for two transductions. Processmore cells in step 10, if necessary.

12. Mix 100 µl of the cells/P1 salts mixture with varying amounts of lysate (1, 10, and100 µl) in sterile test tubes. As a control, include a tube containing 100 µl of cellswithout P1 lysate.

13. Allow phage to adsorb to the cells for 30 min, either at room temperature on thebenchtop or at the appropriate culture temperature. Treat the controls similarly.

Temperature selection should take into account any temperature or cold sensitivity of therecipient strain.

14. Add 1 ml LB broth plus 200 µl of 1 M sodium citrate.

The sodium citrate chelates the calcium essential for phage adsorption to the bacteria,thus minimizing secondary infection by residual phage.

15. Incubate ∼1 hr at 30◦ to 37◦C with aeration.

The exact temperature will depend on the characteristics of the strain being transducedand the expected characteristics of the strain after transduction. If the desired strain istemperature sensitive, the incubation should be done at a low temperature.


1.17.4


16. Centrifuge each culture in a 1.5-ml microcentrifuge tube 2 min at maximum speed,room temperature, to pellet the cells. Remove and discard the supernatant.

17. Suspend the cells in 50 to 100 µl of an appropriate buffer or broth and spread theentire suspension onto the appropriate selective plate. Also spread 100 µl of theP1 stock on another plate. Incubate the plates overnight at the appropriate culturetemperature.

It is good policy to supplement the selective plates with 5 mM sodium citrate, whichhelps prevent readsorption of residual P1 phage. If sodium citrate is used, one-half theconcentration of tetracycline must be used.

Antibiotic resistance is a commonly selected marker: in this case, LB broth can be used fordilution. Be sure to use plates with the lower drug concentrations needed for single-copygenes. Prototrophy can also be selected: in this case, the cells should be washed anddiluted in a minimal salts medium such as TM buffer.

The P1-only control is used to determine whether contaminating bacteria are present inthe lysate.

18. The next day score the plates.

The control plates with cells only or P1 phage only should be free of bacterial colonies;the other plates should contain the transduced colonies. Usually the plate receiving thelowest amount of P1 lysate will have the fewest colonies.

If colonies are present on the cells-only control plate, either the selection is not workingor contamination is present.

If colonies are present on the phage-only plate, the P1 lysate is likely contaminated withdonor bacteria. This should not occur if the CHCl3 treatment was complete. If this controlgives colonies, filter the lysate through a sterile 0.45-µm filter and try again.

19. From the plate that shows growth from the least amount of P1 phage, pick singlecolonies, streak to isolate single clones, and incubate plates overnight at 30◦ to 37◦C.Repeat this process a second time.

Picking colonies from the plate to which the least amount of P1 was added is goodmicrobial practice because many infective P1 phage particles will remain on the plates.Two rounds of purification are essential to ensure removal of such residual P1 phage andseparation of nontransduced bacteria from the actual transductants.

Table 1.17.1 Correlation of Linkage with PhysicalDistance for P1 Transduction

Percentage linkage Physical distance (kb)

0 —

2 67.03

10 49.30

20 38.20

30 30.41

40 24.21

50 18.98

60 14.40

70 10.31

80 6.59

90 3.17

100 0.00


1.17.5


If the genetic marker selected is identical to the mutation of interest (100% linkage), everycolony should express the mutant phenotype. If the genetic marker selected is not tightlylinked to the mutation, more colonies must be screened for the desired phenotype (seeTable 1.17.1).

20. Confirm the genotype of the purified transductants by drug-resistance or phenotype.

If there is no genetic way to score the desired mutation, use PCR to amplify the region ofinterest (using standard primers) and sequence the PCR product.

SUPPORTPROTOCOL

TITRATION OF THE P1 LYSATE

Although P1 transduction is usually performed without titering the phage lysate, thephage should be assayed if the transduction is not successful. This protocol describesspot titering, where the lysate is serially diluted and applied to a bacterial lawn. Spottitering conserves materials and may facilitate visualization of the P1 plaques, which arevery tiny.

Additional materials (also see Basic Protocol)

Standard E. coli K12 strain (e.g., C600 or MG1655)LB plates and top agar (UNIT 1.1), each containing 2.5 mM CaCl2

1. Inoculate 5 ml LB broth with a standard E. coli K12 strain. Incubate overnight untilcells reach mid-log phase (∼2 × 108 cells/ml).

2. Add 1 M CaCl2 to a final concentration of 5 mM.

3. While the cells are growing, make serial ten-fold dilutions (through 10−8) of thephage stock in TM buffer.

4. Pour a lawn of the cells on an LB/CaCl2 plate by mixing 0.25 ml of the cell culturewith 2.5 ml LB top agar (with CaCl2) in a small culture tube and immediately pouringthe contents onto the plate. Tilt the plate gently to distribute the cell-agar mixtureuniformly across the surface of the plate.

5. After the agar hardens, spot 10 µl of each phage stock dilution onto the lawn of cells.Let the spots dry and then incubate the plate upright at 37◦C.

After overnight incubation, individual countable plaques should be present in one of thedilutions.

6. Calculate the titer of infective phage particles/ml (pfu/ml) by counting the numberof plaques and multiplying by the fold dilution and a factor of 100 (to account forthe 10 µl volume spotted).

If the only P1 lysate available has a poor titer (<109 pfu/ml), a new high-titer stock mustbe grown. In this case, use a confluent 10-µl spot of phage to inoculate a liquid cultureand make a high-titer liquid lysate (see Basic Protocol, steps 1 to 8). The spot can betaken from the lawn of cells using a sterile spatula (do not dig into the bottom agar).

ALTERNATEPROTOCOL

PREPARATION OF P1 PLATE LYSATES

Plate stocks can give higher titers than liquid stocks, although they are often not necessary.This is a standard procedure for making P1 plate lysates. Variations exist and may alsowork well. For materials, see Basic Protocol.

1. Inoculate 5 ml LB broth with the E. coli donor strain. Incubate overnight at theappropriate temperature.

The optimal temperature is 37◦C, unless a temperature-sensitive strain is used.Temperature-sensitive strains should be cultured at 30◦ to 32◦C.


1.17.6


2. Dilute the overnight donor culture by inoculating 100 µl into 5 ml LB broth containing0.1% glucose. Incubate ∼1.5 hr until cells reach mid-log phase (∼2 × 108/ml).

Use either a 50-ml Erlenmeyer flask in a shaking water bath or a standard culture tubewith adequate aeration in a test-tube roller.

3. Add 1 M CaCl2 to a final concentration of 5 mM.

4. In a standard 18 × 150–mm glass culture tube, mix 3 ml culture with 50 µl of a 109

to 1010 pfu/ml P1vir stock.

5. Incubate the phage/cell mixture 20 min at 37◦C or room temperature for the phageto adsorb.

6. Add 7.5 ml melted LB top agar to the phage/cell mix and distribute it among threefresh LB plates supplemented with 0.1% glucose and 2.5 mM CaCl2. As a control,pour a plate containing cells alone. Incubate the plates upright overnight.

Do not invert the plates because the top agar is very soft.

Following incubation, the bacterial lawn will appear lysed and may contain mucoidcolonies. Compare the appearance of the control lawn to the plate lysates. A smoothbacterial lawn is an indication that the phage lysate did not grow well.

7. To harvest lysates, scrape the top agar from each plate into a 35-ml centrifuge tube.Wash each plate with 2 ml LB broth and add to the tubes.

8. Add a few drops of CHCl3 to each tube and vortex hard for 30 sec. Let the tubesstand on the bench for 10 min.

9. Centrifuge the tubes 10 min at ∼14,500 × g, 4◦C.

10. Decant the supernatant into a labeled screw-cap tube and store the lysate up to severalyears at 4◦C.

The lysate can be stored for several years at 4◦C and still retain adequate titer. If in doubt,either spot titer as described in the Support Protocol or just make a new lysate.

COMMENTARY

Background InformationThe use of P1 as a transducing phage was

first reported in the early days of molecularbiology (Lennox, 1955), and it has become astandard tool for microbial genome manipu-lation. P1 is a generalized transducing phage,meaning that it will package, and is thus capa-ble of transducing, the entire E. coli genome,although it may have preferred sites in thebacterial DNA where packaging is initiated.After initiation of packaging somewhere onthe bacterial chromosome, the DNA is encap-sidated processively by a headful mechanism.Transducing particles conservatively make up∼0.1% of the total number of phage particlesin a lysate (Yarmolinsky and Sternberg, 1988),and ∼0.1% to 1% of these will contain the par-ticular DNA one wants to move into anotherbacterial strain. The capacity of the P1 headis ∼100 kb; thus ∼100 kb of contiguous bac-terial DNA can be moved from one bacterialstrain to another.

Other P1 phage types have been used fortransduction, for example: P1kc, which formsclear plaques (Lennox, 1955); P1Tn9clr100,a P1 derivative with a chloramphenicol re-sistance gene and a temperature-sensitiverepressor (Silhavy et al., 1984); andhyper-transducing variants (created by NatSternberg), which package the bacterial DNAmore frequently (D. Friedman, pers. comm.).However, all these variants can generate se-rious problems because they are capable oflysogeny. In contrast to phage λ, phage P1lysogens maintain the phage chromosome as aplasmid rather than integrated into the bacte-rial chromosome.

P1 transduction is especially useful in con-junction with the recently developed techniqueof recombineering (UNIT 1.16), a method forin vivo genetic engineering in E. coli. Thetechnique of recombineering allows creationof nearly any desired mutation or genetic con-struct. Providing there is a selection method


1.17.7


for the construct, a genetic construct can becreated by recombineering and then movedinto a different genetic background by P1transduction.


Microbiological methodologyP1 transduction is a genetic procedure

requiring sterile microbiological methods.Glassware and solutions must be sterile, andcare should be used throughout this procedureto avoid contamination. Transductants mustbe purified selectively two consecutive times,preferably on plates containing sodium citrate,to ensure elimination of residual P1 phage. Thecitrate chelates calcium important for efficientphage adsorption to the bacteria, thus mini-mizing infection by the remaining phage.

P1 stockA reasonable P1 stock titer is 109 to

1010 plaque-forming units (pfu)/ml. A freshstock should be obtained from a known sourcebefore beginning this procedure. It is goodpractice to use a P1 stock grown on a wild-type E. coli strain to ensure a high-titer lysate.However, any P1 lysate of good titer can beused to generate a new lysate for transduc-tion, because bacterial DNA present in thefirst lysate is not replicated and will not con-taminate the second lysate. Most investigatorsrarely titer P1 stocks; however, if the transduc-tion is not successful, the phage stock shouldbe titered because a low titer will prevent iso-lation of transduced colonies. Once made, thestock will probably be good for at least a yearif it is not stored over CHCl3, and older stockscan be used if they maintain titer. Storage inthe presence of CHCl3 will cause the titers todrop appreciably within a few months.

LinkageThe closeness of two markers is indicated

by the frequency (as a percentage) of their co-inheritance (see Table 1.17.1). Often the alleleto be transduced is linked to an antibiotic re-sistance gene, and drug resistance is selected.Prototrophy for a metabolic gene can also beselected, e.g., the ability to grow in the absenceof an amino acid. In both cases, the presenceof the second allele must later be confirmedby phenotype or sequencing. Recombineering(UNIT 1.16) can be used to insert a selectablemarker near a gene or mutation that cannotreadily be selected; P1 transduction can thenbe used to move the drug marker and the linkedmutation into a different strain. The laboratory

of Carol Gross has created a set of bacterialstrains with drug insertions at about 1-min in-tervals around the E. coli chromosome (Singeret al., 1989). A collection of E. coli single-gene knockout mutants (the Keio collection)has also recently been described (Baba et al.,2006). P1 can be grown on any of these strainsand transduction used to move the drug markerin a particular region of the genome into an-other more desirable background.

Host elements that interfere withtransduction

Once a recipient strain has been infectedwith the P1 lysate, incorporation of the bac-terial DNA into the recipient chromosomerequires host recombination functions, espe-cially RecA and RecBCD. It is thus not pos-sible to transduce a recA mutant strain, sinceabsence of the RecA function reduces homol-ogous recombination ∼1000-fold. Mutationsin the RecBCD enzyme also affect this pro-cedure adversely, although these mutants areless defective for recombination than are recAmutants. To use P1 transduction to build anE. coli strain with multiple mutations, includ-ing one in recA, the recA mutation shouldbe incorporated last. These recombination-defective strains are also poor hosts for phageP1 and give lower-titer lysates. Mutationsin other recombination functions may alsohave a detrimental effect. At least one non-recombination-related mutation also impactsP1 growth. Phage P1 adsorbs to a sugar residueof the lipopolysaccharide (LPS) on the E. colicell surface (Sandulache et al., 1984), and amutation in the galU gene, which encodes acomponent of the LPS, makes E. coli unableto support the growth of phage P1 (Franklin,1969).

Be extremely careful of using any P1 stockmade on bacterial lysogens containing phagessuch as λ or Phi80. These other phage typeswill be present in the P1 stock and can causeserious contamination problems when succes-sive P1 stocks are made from them. An ex-ception is the defective λ prophage present inthe recombineering strains referred to in UNIT

1.16. Since this prophage is incapable of exci-sion from the bacterial chromosome, it cannotcontribute contaminating λ phage particles.

Anticipated ResultsSince one in ∼103 phages in a P1 lysate are

transducing phage, and phage P1 can package1% of the E. coli chromosome, each 1% of thebacterial chromosome will be represented byone transducing phage in a population of 105

total phage. Since a high-titer lysate contains


1.17.8


∼109 phages/ml, and since the frequency ofincorporation into the recipient by recombina-tion will be <100%, for any one marker typ-ically there will be ∼100 to 1000 successfultransduction events/ml of lysate. This roughcalculation assumes equal transduction of allparts of the E. coli chromosome, although inreality some regions of the genome are “hot-ter” and will transduce better than others.

Time ConsiderationsThe entire procedure of growing the

overnight cultures, making the P1 lysate, per-forming the transduction, and purifying thecolonies will take less than a week. Once thelysate is grown and the transduction is com-plete, the steps on subsequent days are simpleand should only take a few minutes of actualhands-on time.

Literature CitedBaba, T., Ara, T., Hasegawa, M., Takai, Y., Oku-

mura, Y., Baba, M., Datsenko, K. A., Tomita, M.,Wanner, B. L., and Mori, H. 2006. Constructionof Escherichia coli K-12 in-frame, single-geneknockout mutants: The Keio collection. Mol.Syst. Biol. 2:2006.0008.

Franklin, N.C. 1969. Mutation in gal U gene of E.coli blocks phage P1 infection. Virology 38:189-191.

Lennox, E.S. 1955. Transduction of linked geneticcharacters of the host by bacteriophage P1. Vi-rology 1:190-206.

Sandulache, R., Prehm, P., and Kamp, D. 1984.Cell wall receptor for bacteriophage Mu G(+).J. Bacteriol. 160:299-303.

Silhavy, T.J., Berman, M.L., and Enquist, L.W.1984. Preparation of a P1Tn9clr100 lysate. In

Experiments with Gene Fusions, pp. 109-110.Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

Singer, M., Baker, T.A., Schnitzler, G., Deischel,S.M., Goel, M., Dove, W., Jaacks, K.J., Gross-man, A.D., Erickson, J.W., and Gross, C.A.1989. A collection of strains containing geneti-cally linked alternating antibiotic resistance ele-ments for genetic mapping of Escherichia coli.Microbiol. Rev. 53:1-24.

Yarmolinsky, M.B. and Sternberg, N. 1988. Bacte-riophage P1. In The Bacteriophages, Vol. 1. (R.Calendar, ed.) pp. 291-438. Plenum Press, NewYork.

Key ReferencesMiller, J.H. 1992. A Short Course in Bacterial Ge-

netics: A Laboratory Manual and Handbookfor Escherichia coli and Related Bacteria. ColdSpring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Provence, D.L. and Curtiss, R. III. 1994. Gene trans-fer in gram-negative bacteria. In Methods forGeneral and Molecular Bacteriology (P. Ger-hardt, R.G.E. Murray, W.A. Wood, and N.R.Krieg, eds.) pp. 317-347. ASM Press, Washing-ton, D.C.

Silhavy, T.J., Berman, M.L., and Enquist, L.W.1984. Experiments with Gene Fusions. ColdSpring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

These are classical references for transduction byphage P1 and other phages.

Internet Resourceshttp://rothlab.ucdavis.edu/protocols/

p1-transduction-chart.htmlThis Web site contains a more complete version ofTable 1.17.1.

Documents

sun-lab.med.nyu.edusun-lab.med.nyu.edu/files/sun-lab/attachments/CPMB.Ch.01.E coli.pdf · cos site of action of phage lambda ter function. Cos site is cut by ter to yield two cohesive