Competent cells, vectors, media & antibiotics for recombinant protein production Product Guide

7/29/2019 Competent cells, vectors, media & antibiotics for recombinant protein production Product Guide

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Paint your own

protein.With EMD Millipores protein expressionplatorms, you can choose the proteins

you puriy to answer the precise biological

questions youre tackling. Dont let limitations

like solubility, yield, or toxicity inuence

your choice o proteins to study. With the

expertise o Novagen, the household name

in cloning and recombinant protein expression

technology, EMD Millipore makes it possible

to elucidate meaningul unctions o even the

most intractable gene products.


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Contents

4 Competent Cells

8 Cloning Strains

10 Expression Strains

21 Vectors

22 Cloning Vectors

24 Expression Vectors

34 Culture Media

35 Molecular Biology Media

38 Insect Cells and Culture Media

39 Protein Production inSuspension Cell Systems

40 NovaCHOice Transection Kit

41 Antibiotics


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Competent CellsEMD Millipores competent cells include the widest variety o

protein expression strains available, as well as both undamental

and advanced strains or cloning applications. Whether you have

a well-behaved recombinant protein or are acing challenges

with truncation, insolubility, or toxicity, and whether you need a

trustworthy strain or routine cloning or a specialized strain or

efcient library construction, our cells can move your research

orward.

Since reliability is critical, we veriy the phenotype and purity

o each strain and guarantee its transormation efciency. With

years o experience in producing competent cells or protein and

molecular biology research, we provide robust perormance you

can count on.

Our competent cells are available in ormats ranging rom

standard 0.2 mL aliquots to convenient 50 L Singles Compe-

tent Cells. Doing hundreds to thousands o transormations at

a time? We oer strains in HT96 ormat or high-throughput

applications. Need cells that completely lack animal materials?

Animal-ree Veggie Competent Cell versions are available or

several strains.

I.


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Features Guaranteed transormation efciencies o

>2 106 cu/mg

Provided as rozen single-use aliquots in 11-reaction or22-reaction kits

Includes Test Plasmid and SOC Medium

Selection DE3 lysogens or protein expressionwith pET vectors

Prepared using an optimized chemical method

Features Guaranteed efciency o > 2 106 cu/mg

Manuactured ree o animal-derived mediaand components

Easy-to-use Singles ormat

Prepared using an optimized chemical method

Defcient in lon and ompTproteases

Competent Cell Kit Confgurations

Kit Component Standard Kits Singles Competent Cells HT96 Competent Cells

0.4 mL 1 mL 11 reactions 22 reactions 1 plate 4 plates

Competent Cells 2 x 0.2 mL 5 x 0.2 mL 11 x 50 L 22 x 50 L 96 x 20 L 4 x (96 x 20 L)

Test Plasmid 10 L 10 L 10 L 10 L 10 L 2 x 10 L

SOC Medium* 2 x 2 mL 4 x 2 mL 2 x 2 mL 4 x 2 mL 14 mL 4 x 14 mL

8-cap Strip - - - - 1 pkg o 12 4 pkgs o 12

Reagent Reservoir - - - - 1 4

HT96 Lids - - - - 1 4

* The SOC Medium included in Veggie Competent Cells is an animal-ree preparation.

Protein expression using VeggieSingles competent cells andVeggie media components

Veggie BL21(DE3) was transormedwith two constructs, grown in TBmedium made with Veggie Peptone andVeggie Yeast Extract, and induced or 3hours with 1 mM IPTG. Samples o totalcell protein were analyzed by SDS-PAGEand Coomassie blue staining.

Singles Competent Cell Format

Veggie Competent Cells in Singles Format

Singles Competent Cells are designed or ultimate

convenience and reliability in plasmid transormation.

Cells are provided in 50-L volumes, eliminating the

need to aliquot, reeze/thaw, or waste partially used

vials, saving time and money and ensuring reliable cellperormance. To use, simply thaw, add DNA, incubate 5

minutes on ice, heat shock or 30 seconds, place on ice

or 2 minutes, and add SOC medium.

All Veggie Competent Cells are maintained and

manuactured with media and reagents derived rom

non-animal sources, making these cells ideally suited

or applications in which animal-ree materials are nec-

essary. Cells are provided in 50-mL volumes, eliminating

the need to aliquot, reeze/thaw, or waste partially used

vials. Each kit includes Test Plasmid and Veggie SOC

Medium.

Lane SampleA pET-32b(+)(Thioredocin)

B pET-41b(+)(GST)

U Uninduced total cell protein

I Induced total cell protein

M Perect Protein Markers,10-225 kDa

kDa225

A B

U UI M I

150

100

75

50

35

25

15

10

Competent Cells


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Competent Cells

Features Guaranteed efciency >1 108 cu/mg

High-throughput, 96-well ormat

HT96 CompetentCells Format

High-efciency competent cells

predispensed in a 96-well plate or

high throughput applications

HT96 Competent Cells are designed or high-through-put transormation. The cells are predispensed in 20-L

volumes in a sturdy 96-well polypropylene plate compat-

ible with a variety o thermal cyclers and water baths.

Wells are individually sealed and have raised rims to

prevent cross-contamination. You can pierce the seals

with standard pipette tips or remove them or easier

access. Strips o caps are also provided or reliable sealing

during manipulation and storage. For processing smaller

numbers o samples, you can easily split groups o 24

wells rom the plate.

HT96 Isothermal Block

Efcient thermal transer to samples

in 96-well plates or high throughput

bacterial transormation

The HT96 Isothermal Block is an anodized aluminum,

solvent-resistant block specically designed to hold oneHT96 plate and to provide ecient thermal transer to

samples held within the 96-well plate. Using an HT96

Isothermal Block or each temperature, you can rapidly

transer samples between the low-temperature and

heat-shock steps in transormation protocols. Simply

preincubate the anodized aluminum block at the desired

temperature and place the HT96 Competent Cell plate in

the block. The HT96 Isothermal Block is compatible with

most 96-well PCR plates and robotic platorms.

Ordering Inormation

Description Size Catalogue No.

HT96 NovaBlue Competent Cells 1 plate 71011-3

4 plates 71011-4

HT96 BL21(DE3) Competent Cells 1 plate 71012-3

4 plates 71012-4

HT96 Isothermal Block 1 each 71195-3

Features Guaranteed efciency >1 108 cu/mg

High-throughput, 96-well ormat
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Competent Cells

Competent Cells Selection Guide

Expression StrainsProblem/Solution Strains

General Protein Expression

Solution: Maximize yields by using host strains decient in Lon and OmpT

proteases

BL21

BL21(DE3)

Insoluble Protein/No Activity

Problem 1: Reduction o disulde bonds/misolded protein

Solution: Minimize protein reduction in cytoplasm; use trxB/gor-

hosts

Origami 2

Origami 2(DE3)

Rosetta-gami 2 Rosetta-gami 2(DE3)

Rosetta-gami B

Rosetta-gami B(DE3)

Problem 2: High levels o expression resulting in misolded protein

Solution: Attenuate expression/ titrate IPTG; use lacY- hosts

Tuner

Tuner(DE3)

Rosetta-gami B

Rosetta-gami B(DE3)

No Protein/Cell Death

Problem: Toxic protein

Solution: Suppress basal expression, use a stringent control host

Tuner

Tuner(DE3)

NovaBlue

NovaBlue(DE3)

Any pLysS host

Certifed Animal-Free

Veggie BL21(DE3)

Veggie BL21(DE3)pLysS

Truncated Protein

Problem: : E. colicodon bias

Solution: Use a host that supplies rare tRNAs

Rosetta(DE3)

Rosetta 2

Rosetta 2(DE3)

Rosetta-gami 2

Rosetta-gami 2(DE3)

Rosetta-gami B

Rosetta-gami B(DE3)

RosettaBlue RosettaBlue(DE3)

Stabilizing Target Plasmids

Problem: Target plasmid unstable due to repetitive sequences

Solution: Use recA- hosts to minimize recombination caused by repeated

sequences

BLR(DE3)

HMS174

HMS174(DE3)

NovaBlue

NovaBlue(DE3)

Methionine Protein Labeling

Solution: Use a methionine auxotroph host or higher specic activity B834

B834(DE3)

Cloning StrainsBlue/White Screening Phage Resistant Certifed Animal-Free

NovaBlue NovaBlue T1R Singles Veggie NovaBlue Singles

NovaBlue GigaSingles

NovaBlue T1R

SinglesVeggie NovaBlue Singles


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Competent Cells

Cloning StrainsFor successul isolation and propagation o engineered DNA vectors, transormation

into competent bacterial cells is crucial. The right strain o competent cells can makethe dierence between cloning rustration and progressing to the next step.

EMD Millipore oers a range o strains and ormats to suit every cloning project.

NovaBlue is a K-12 strain ideally suited as an initial

cloning host due to its high transormation eciency,

blue/white screening capability (with appropriate

plasmids) and recA endA mutations, which result in high

yields o excellent quality plasmid DNA.

NovaBlue T1R strain has the added benet o resistance

to T1 and T5 phage.

NovaBlue Strain Competent Cells

For routine initial cloning, time-tested NovaBlue

Competent Cells are ideal. NovaBlue is a K-12

derivative that oers high transormation eciency,

acilitates plasmid stability, and allows blue/white

screening with appropriate plasmids.

Blue/white screening in NovaBlue SinglesCompetent Cells

A 212 bp DNA ragment was amplifed DNA polymeraseand treated with the Perectly Blunt End Conversion Mix.The insert was ligated to the pETBlue-2 Blunt Vector andtransormed into NovaBlue Singles Competent Cells.Uncut pETBlue-2 plasmid was also transormed in parallel

to serve as a no-insert control. The transormationreactions were plated on LB agar containing 50 g/mLcarbenicillin, X-gal and IPTG, and incubated overnightat 37C.


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Competent Cells

Host Strains Derivation Guaranteed Efciency

(cu/g)

Packaging Format Resistance* Key Feature(s)

& Application

NovaBlue K-12 > 1.5 x 108 Standard, Singles Tet Non-expression2 host, general purpose cloning

host, plasmid preps

NovaBlue K-12 > 1.0 x 108 HT96 Tet Non-expression2 host, high-throughput cloning

NovaBlue

GigaSingle

K-12 > 1.0 x 109 Singles Tet Non-expression2 host, high-eciency cloning

Veggie

NovaBlue

K-12 > 1.5 x 108 Singles Tet Non-expression2 host, general purpose cloning

host, plasmid preps with

non-animal origin components

NovaBlue T1R K-12 > 1.5 x 108 Singles Tet non-expression2 host, general purpose cloning,

plasmid preps, T1 and T5 phage resistant

NovaBlue Genotype:

endA1 hsdR17(rK12-mK12

+) supE44 thi-1 recA1 gyrA96 relA1 lacF[proA+B+

laclqZM15::Tn10] (TetR

)

NovaBlue T1R Genotype:

endA1 hsdR17 (rK12 mK12

+) supE44 thi-1 recA1 gyrA96 relA1 lac tonA

F[proA+

B+

lacIqZM15::Tn10] (TetR

)

Ordering Inormation


NovaBlue Singles Competent Cells 11 reactions 70181-3

22 reactions 70181-4

NovaBlue GigaSingles Competent Cells 11 reactions 71227-3


NovaBlue T1R Singles Competent Cells 11 reactions 71318-3


Veggie NovaBlue Singles Competent Cells 11 reactions 71251-3


HT96 NovaBlue Competent Cells 1 plate 71011-3

4 plates 71011-4
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0 Competent Cells

Expression StrainsWhen expressing recombinant proteins in E. coli, the goals are to obtain high yields

o ull-length, soluble protein. Whether you are producing protein or enzymatic assays,generating antigen or antibody production, assaying protein-protein interactions,

or determining three-dimensional structure you need high-quality protein, ast.

EMD Millipores portolio o bacterial strains or protein expression includes the best

all-purpose strains and several specialty strains or difcult-to-express proteins, all

backed by unwavering technical support to increase your chances o success. For

ultimate convenience and reliability in plasmid transormation, Singles Competent

Cells are provided in 50 L volumes to eliminate the need to aliquot, reeze/thaw, or

waste partially used vials. This saves time, money, and ensures reliable cell perormance.

Variations o Bacterial Expression Strains

Strain Designation Characteristic

(DE3) Host is a lysogen o DE3 and carries a chromosomal copy o the T7 RNA polymerase gene under control o

the lacUV5 promoter. Such strains are required or protein expression rom target genes cloned in T7 expres-

sion vectors (eg. pET vectors).

pLysS Strain carries a plasmid encoding T7 lysozyme, a natural inhibitor o T7 RNA polymerase. Strains suppress

basal expression o T7 RNA polymerase prior to induction and, thereby, stabilize pET, pCDF, pRSF, pACYC-

Duet, pCOLADuet and Gateway Nova pDEST and pCOLADuet recombinants encoding target proteins

that aect cell growth and viability.

pLacI Host strains that carry the pLacI plasmid produce extra Lacrepressor, which is required to

suppress basal expression rom pETBlue and pTriEx vectors.

Non-DE3 Host Strains

Host Strain(s)

Transormation

Efciency

NovaBlue

RosettaBlue

> 1 x 108 cu/g

HMS174 > 5 x 106 cu/g

BL21BLR

Origami 2

Origami B

Rosetta 2

Rosetta-gami 2

Rosetta-gami B

Tuner

> 2 x 106 cu/g

T7 Expression Host Strains

Host Strain(s)

Transormation

Efciency

NovaBlue(DE3)

RosettaBlue (DE3), RosettaBlue (DE3)pLysS

> 1 x 108 cu/g

HMS174(DE3), HMS174(DE3)pLysS > 5 x 106 cu/g

B834(DE3), B834(DE3)pLysS

BL21(DE3), BL21(DE3)pLysS

BLR(DE3), BLR(DE3)pLysS

Origami 2(DE3), Origami 2(DE3)pLysS

Origami B(DE3)

Rosetta (DE3), Rosetta (DE3)pLysS

Rosetta 2(DE3), Rosetta 2(DE3)pLysS

Rosetta-gami 2(DE3), Rosetta-gami 2(DE3)pLysS

Rosetta-gami B(DE3), Rosetta-gami B(DE3)pLysS

Tuner(DE3), Tuner(DE3)pLysS

> 2 x 106 cu/g

Origami B(DE3)pLysS > 1 x 106 cu/g


11/44

1Competent Cells

Expression Host Strain Competent Cell Set Selection Guide

BL21

BL21(DE3)

BL21(DE3)pLysS

BLR(DE3)

BLR(DE3)pLysS

HMS174

HMS174(DE3)

HMS174(DE3)pLysS

NovaBlue

NovaBlue(DE3)

Origami

2

Origami

2(DE3)

Origami

2(DE3)pLysS

Origami

B

Origami

B(DE3)

Origami

B(DE3)pLysS

Rosetta

2

Rosetta

2(DE3)

Rosetta

2(DE3)pLysS

RosettaBlue

RosettaBlue(DE3)

RosettaBlue(DE3)pLysS

Rosetta-gami

2

Rosetta-gami

2(DE3)

RRosetta-gami

2(DE3)pLy

sS

Rosetta-gami

B

Rosetta-gami

B(DE3)

Rosetta-gami

B(DE3)pLys

S

Tuner

Tuner

(DE3)

Tuner(DE3)pLysS

Cat. No.

Non-DE3 Competent Cell Set 1 71211-3

(DE3) Competent Cell Set 1 71207-3

(DE3) Competent Cell Set 2 71208-3

(DE3)pLysS Competent Cell Set 1 71209-3

(DE3)pLysS Competent Cell Set 2 71210-3

BL21 Competent Cell Set 70232-3

HMS174 Competent Cell Set 70234-3Origami 2 Competent Cell Set 71431-3

Origami B Competent Cell Set 70911-3

Rosetta 2 Competent Cell Set 71405-3

RosettaBlue Competent Cell Set 71079-3

Rosetta-gami 2 Competent Cell Set 71432-3

Rosetta-gami B Competent Cell Set 71177-3

Tuner Competent Cell Set 70726-3

Contains 2 0.2 mL cells Contains 0.2 mL cells

General Protein ExpressionBL21 has been the gold standard or protein expression since it was frst introduced in

1990. Defcient in lon and ompTproteases, BL21 and its derivatives are high-yielding

and ideal or many applications.

TCP TCP PC PC PC PC

plysS plysS lysS lysS

pET-28b()b-gal

M Perect Protein Markers

TCP Total Cell Protein

PC PopCulture extract

lys chicken lysozyme

lys recombinant lysozyme

pLysS BL21 (DE3) pLysS host

b-gal

GST

Using a pLysS host strain increases theefciency o protein extraction.

BL21(DE3) and BL21(DE3)pLysS hostscontaining pET b-galactosidase recombinantplasmids were growin in liquid culture and

protein expression induced with 1 mM IPTGor 3 h. Samples o cultures were extractedusing PopCulture reagent, with the indicatedlysozyme treatments. Total cell protein (TCP)samples were prepared by resuspending cellpellets in SDS sample buer. The TCP andequal volumes o all PopCulture extractswere analyzed by SDS-PAGE (4-20% gradientgels) and Coomassie blue staining.
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12/44

2 Competent Cells

Host Strains Derivation Guaranteed Efciency Packaging Format Resistance*

Key Feature(s)

& Application

BL21 B834 > 2.0 x 107 Standard none Routine protein expression, control

non-expression2 host

BL21(DE3) B834 > 2.0 x 107 Standard, Singles,

HT96

none General purpose expression3 host

BL21(DE3)pLysS B834 > 2.0 x 107 Standard, Singles Cam High-stringency expression 3, 4

Veggie

BL21(DE3)

B834 > 2.0 x 107 Singles none Protein expression that requires material ree

o animal origin

Veggie

BL21(DE3)pLysS

B834 > 2.0 x 107 Singles Cam High-stringency expression 3, 4, ree o

animal origin

BL21(DE3) Genotype:

F ompT hsdSB(rB mB

) gal dcm (DE3)

BL21(DE3)pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm (DE3) pLysS (CamR)

Ordering Inormation


BL21(DE3) Singles Competent Cells 11 reactions 70235-3


BL21(DE3) pLysS Singles Competent Cells 11 reactions 70236-3


Veggie BL21(DE3) Singles Competent Cells 11 reactions 71252-3


Veggie BL21(DE3) pLysS Singles Competent Cells 11 reactions 71253-3


HT96 BL21(DE3) Competent Cells 1 plate 71012-3

4 plates 71012-4

BL21 Competent Cells Set 1 set 70232-3

Enhance disulfde bond ormation incytoplasm with Origami 2 andOrigami B Strain Competent Cells

Origami 2 and Origami B strains have mutations in glu-

tathione reducatase (gor) and thioredoxin reductase (trxB),

acilitating proper disulde bond ormation. These strains

also include the lon and ompTdeciencies o BL21, which

increase protein stability. Using Tuner competent cells to

nely control IPTG concentration is another approach to

acilitate solubility.

Origami 2 host strains are K-12 derivatives that havemutations in both the thioredoxin reductase (trxB)and

glutathione reductase (gor)genes, which greatly enhance

disulde bond ormation in the cytoplasm. The Origami

2 strains are kanamycin sensitive, making these host

strains compatible with many Novagen expression

vectors. The gormutation is still selected or by

tetracycline.

Origami B host strains carry the same trxB/gormutations as the original Origami strain, except that they

are derived rom a lacZYmutant o BL21 to enable precise

control o expression levels by adjusting the concentration

o IPTG. Thus the Origami B strains combine the desirable

characteristics o BL21, Tuner, and Origami hosts in one

strain background. The trxBand gormutations are selecta-

ble on kanamycin and tetracycline, respectively; thereore,

these strains are not compatible with kanamycin- or

tetracycline-resistant plasmids.

Insoluble Protein/No ActivityTo reduce the possibility o disulfde bond ormation between molecules, strains

containing mutations in trxBand gorare recommended only or the expression

o proteins that require disulfde bond ormation or proper olding.
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13/44

1Competent Cells

Host Strains Derivation

Guaranteed Efciency

(cu/g)

Packaging

Format Resistance* Key Feature(s) & Application

Origami 2 K-12 > 2.0 x 106 Standard Tet + Str control non-expression2 host; kanamycin

sensitive

Origami 2(DE3) K-12 > 2.0 x 106 Standard,

Singles

Tet + Strt general expression3 host; two mutations

in cytoplasmic disulde reduction pathway

enhance disulde bond ormation in E. coli

cytoplasm; kanamycin sensitive

Origami 2(DE3)

pLysS

K-12 > 2.0 x 106 Standard Tet + Str+ Cam high-stringency3,4 expression host; two

mutations in cytoplasmic disulde reductionpathway enhance disulde bond ormation

in E. colicytoplasm; kanamycin sensitive

Origami 2(DE3)

pLacI

K-12 > 2.0 x 106 Standard Tet + Str+ Cam

Origami B Tuner

(B strain)

> 2.0 x 106 Standard Kan + Tet control non-expression2 host

Origami B(DE3) Tuner

(B strain)

> 2.0 x 106 Standard Kan + Tet general expression3 host; contains Tuner lac

permease mutation and trxB/gormutations

or cytoplasmic disulde bond ormation

Origami B(DE3)

pLysS

Tuner

(B strain)

> 2.0 x 106 Standard Kan + Tet + Cam high-stringency3,4 expression host; contains

Turner lacpermease mutation and

trxB/gormutations or cytoplasmic

disulde bond ormationOrigami

B(DE3)pLacI

Tuner

(B strain)

> 2.0 x 106 Standard Kan + Tet + Cam BL21 lacZYdeletion mutant; allows precise

control with IPTG

Origami 2 Genotype:

(ara-leu)7697 lacX74 phoA PvuII phoR araD139 ahpC galE galK rpsL

F[lac+ lacIqpro] gor522::Tn10 trxB(StrR, TetR)

Origami 2(DE3) Genotype:


F[lac+ lacIqpro] (DE3) gor522::Tn10 trxB(StrR, TetR)

Origami 2(DE3)pLysS Genotype:


F[lac+ lacIqpro] (DE3) gor522::Tn10 trxB pLysS(CamR, StrR, TetR)

Origami 2(DE3)pLacI Genotype:

(ara-leu)7697 lacX74 phoA PvuII phoR araD139 ahpC galE galK rpsLF[lac+ lacIqpro] (DE3) gor522::Tn10 trxB pLacI(CamR, StrR, TetR)

Origami B Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 ahpC gor522::Tn10 trxB(KanR, TetR)

Origami B(DE3) Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 ahpC gor522::Tn10 trxB(KanR, TetR)

Origami B(DE3)pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 ahpC gor522::Tn10 trxBpLysS (CamR,

KanR, TetR)

Origami B Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 ahpC gor522::Tn10 trxBpLacI (CamR,

KanR, TetR)

Ordering Inormation


Origami 2(DE3) Singles Competent Cells 11 reactions 71408-3


Origami 2(DE3)pLysS Competent Cells 0.4 mL 71345-3

1 mL 71345-4

Origami 2(DE3)pLacI Competent Cells 0.4 mL 71347-3

Origami B(DE3) Competent Cells 0.4 mL 70837-31 mL 70837-4

Origami B(DE3)pLysS Competent Cells 0.4 mL 70839-3

1 mL 70839-4

Origami B(DE3)pLacI Competent Cells 0.4 mL 70838-3

1 mL 70838-4

Origami 2 Competent Cells Set 1 set 71431-3

Origami B Competent Cells Set 1 set 70911-3
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14/44

4 Competent Cells

Overcome E. colicodon bias withRosetta & Rosetta 2StrainCompetent Cells

Rosetta and Rosetta 2 host strains are BL21 deriva-

tives designed to enhance the expression o eukaryotic

proteins that contain codons rarely used in E. coli. The

original Rosetta strains supply tRNAs or the codons

AUA, AGG, AGA, CUA, CCC, and GGA on a compatible

Lane Sample

M Perect Protein Markers,

15-150k Da

C Crude extract

P Purifed target protein

HisTag/BRCA2

T7Tag/Rad51

HisTag/Rad51

15

25

35

50

75

100

150

kDa

Protein B:

Protein A:

M 1

C

+

T7Tag/

Rad51

HisTag/

BRCA2

HisTag/

Rad51

HisTag/

BRCA2

2

P

3

C

4

P

5

C

6

P

Lane Sample

1 Rosetta (DE3) total cell

extract (induced)

2 Rosetta 2(DE3) total cell

extract (induced)

M Perect Protein Markers,

10-225 kDa

b-gal (5 x CGG mutant)

10

15

25

35

50

75

100150225

kDa

M1 2

Using Rosetta(DE3) competent cells orcoexpression o interacting domains oBRCA2 and Rad51.

Three constructs, pET-30 Ek/LIC BRCA2Rad51, pET-30 Ek/LIC BRCA2, and pET-30 Ek/LIC Rad51, were transormed intoRosetta(DE3) competent cells, grown inLB broth, and induced with IPTG at 26Cor 4 h. Cells were harvested by centrigationand lysed with BugBuster Protein Extrac-

tion Reagent, rLysozyme Solution, andBenzonase Nuclease. Equal volumes werepuriedbyNi-NTAHisBindchromatographyunder native conditions. Samples representingequal cell mass were analyzed by SDS-PAGE(420% gradient) and stained withCoomassie blue.

Rosetta 2(DE3) cells enhance

expression o a protein containingconsecutive CGG rare codons.

A pET-15b recombinant plasmid containingfve consecutive CGG codons near the 5-end o the b-gal coding region wastransormed into Rosetta(DE3) and Rosetta2(DE3). Cells were grown in LB broth withcarbenicillin and chloramphenicol to an OD600between 1.0 and 1.2, induced with 1 mMIPTG (3 hours at 37C), and harvestedby centriugation. Cells were resuspendedand lysed in SDS sample buer, ollowed bysonication to reduce sample viscosity. Proteinswere separated on a 420% SDS polyacryl-amide gel and stained with Coomassie blue.

Truncated ProteinI youre studying an eukaryotic protein, its ORF may well contain codons

that are rarely employed in E. coli. Rosettaand Rosetta2 strains include

a chloramphenicol-selectable plasmid bearing tRNAs or codons that are

inrequently used in E. coli, thus conerring universal translation.

chloramphenicol-resistant plasmid, pRARE. The Rosetta

2 strains supply a seventh rare codon (CGG) in addition to

the six ound in the original Rosettastrains. By supplying

rare codons, the Rosetta strains provide or universal

translation, where translation would otherwise be limited

by the codon usage oE. coli. The tRNA genes are driven by

their native promoters. In the pLysS and pLacI derivatives

o these strains, the rare tRNA genes are present on the

same plasmids that carry the T7 lysozyme and lac

repressor genes, respectively.


15/44

1Competent Cells


(cu/g)

Packaging

Format

Resistance* Key Feature(s)

& Application

Rosetta BL21 > 2.0 x 106 Standard Cam control non-expression2 host

Rosetta(DE3) BL21 > 2.0 x 106 Standard Cam general expression3 host; provides six rare

codon tRNAs

Rosetta(DE3)

pLysS

BL21 > 2.0 x 106 Standard Cam high-stringency3, 4 expression host; provides

six rare codon tRNAs

Rosetta(DE3)

pLacI

BL21 > 2.0 x 106 Standard Cam

Rosetta 2 BL21 > 2.0 x 106 Standard Cam control non-expression2 host

Rosetta 2(DE3) BL21 > 2.0 x 106 Standard,

Singles

Cam general expression3 host; provides seven rare

codon tRNAs

Rosetta 2(DE3)

pLysS

BL21 > 2.0 x 106 Standard,

Singles

Cam high-stringency3, 4 expression host; provides

seven rare codon tRNAs

Rosetta 2(DE3)

pLacI

BL21 > 2.0 x 106 Standard Cam

Rosetta Genotype:

FompT hsdSB(rB mB

) gal dcm pRARE(CamR)

Rosetta (DE3) Genotype:

F ompT hsdSB(rB mB

) gal dcm (DE3) pRARE(CamR)

Rosetta (DE3)pLysS Genotype:F ompT hsdSB(rB

mB) gal dcm (DE3)pLysSRARE (CamR)

Rosetta (DE3)pLacI Genotype:

F ompT hsdSB(rB mB

) gal dcm (DE3)pLacIRARE(CamR)

Rosetta 2 (DE3)pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm pLysSRARE2 (CamR)

Rosetta 2 (DE3)pLacI Genotype:F ompT hsdSB(rB

mB) gal dcm (DE3)pLacISRARE2(CamR)

Ordering Inormation


Rosetta (DE3) Competent Cells 0.4 mL 70954-3

1 mL 70954-4

Rosetta (DE3)pLysS Competent Cells 0.4 mL 70956-3

1 mL 70956-4

Rosetta (DE3)pLacI Competent Cells 0.4 mL 70920-3

1 mL 70920-4

Rosetta 2(DE3) Singles Competent Cells 11 reactions 71400-3


Rosetta 2(DE3) pLysS Singles Competent Cells 11 reactions 71401-3


Rosetta 2(DE3)pLacI Competent Cells 0.4 mL 71404-3

1 mL 71404-4

Rosetta 2 Competent Cells Set 1 set 71405-3
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16/44

6 Competent Cells

E. colicodon biasDisulfde bond ormationUnstable target plasmids

Solve common recombinant protein expression issues with Rosetta-gami 2,Rosetta-gami B, & RosettaBlue Strain Competent Cells hybrid strains.

The Rosetta-gami 2, Rosetta-gami B, and RosettaBlue host strains are

Rosetta or Rosetta 2 hybrid strains designed to enhance the expression o

eukaryotic proteins that contain codons rarely used in E. coli. Additionally,

each hybrid strain also oers another unique beneft to overcome common

recombinant protein expression issues.

Rosetta-gami 2 strainsThese host strains combine eatures o Origami 2

and Rosetta 2, allowing or enhanced disulde bond

ormation and enhanced expression o eukaryotic proteins

that contain codons rarely used in E. coli. These strains

are derived rom Origami 2, a kanamycin-sensitive K-12

strain carrying the trxBand gormutations or disulde

bonds ormation in the cytoplasm. The cells carry the

chloramphenicol-resistant plasmid, pRARE2, which

supplies tRNAs or seven rare codons, AUA, AGG, AGA,

CUA, CCC, GGA, and CGG under the control o their native

promoter. The gormutation is selectable on tetracycline.

Rosetta-gami B strainsRosetta-gami B strains combine the key eatures o BL21

(and its Tuner derivative), Origami, and Rosetta to

enhance both the expression o eukaryotic proteins and

the ormation o target protein disulde bonds in the

bacterial cytoplasm. These strains are compatible with

ampicillin- or spectinomycin-resistant vectors.

RosettaBlue strainsThese are NovaBlue derivatives that combine high

transormation eciency and recA, endA and lacIq

mutations with enhanced expression o eukaryotic

proteins that contain codons rarely used in E. coli. These

strains supply tRNAs or AGG, AGA, AUA, CUA, CCC, and

GGA on a compatible chloramphenicol-resistant plasmid.

In RosettaBlue(DE3)pLysS and RosettaBlue(DE3)pLacI,

the rare tRNA genes are present on the same plasmids

that carry the T7 lysozyme and lacrepressor genes,

respectively. Blue/white screening is not possible with

RosettaBlue(DE3) strains due to the presence o the lacZ-peptide coding sequence in the DE3 lysogenic phage.


17/44

1Competent Cells


(cu/g)

Packaging

Format


& Application

Rosetta-gami 2 Origami 2

(K-12)

> 2.0 x 106 Standard Tet + Str + Cam Expresses seven rare tRNAs; acil itatesexpression o genes that encode rare E.colicodons

Kan sensitive, trxB/gormutant, greatlyacilitates cytoplasmic disulde bondormation, Leu auxotroph

Rosetta-gami

2(DE3)

Origami 2

(K-12)

> 2.0 x 106 Standard Tet + Str + Cam

Rosetta-gami

2(DE3)pLysS

Origami 2

(K-12)


Rosetta-gami2(DE3)pLacI

Origami 2(K-12)


Rosetta-gami B Origami B

(B strain)

> 2.0 x 106 Standard Kan + Tet + Cam Expresses six rare tRNAs; acilitatesexpression o genes that encode rare E.colicodons

trxB/gormutant, greatly acilitatescytoplasmic disulde bond ormation

BL21 lacZYdeletion mutant; allows precise

control with IPTG

Rosetta-gami

B(DE3)

Origami B

(B strain)

> 2.0 x 106 Standard Kan + Tet + Cam

Rosetta-gami

B(DE3)pLysS

Origami B

(B strain)


Rosetta-gami

B(DE3)pLacI

Origami B

(B strain)


RosettaBlue Origami 2

(K-12)

> 1.0 x 106 Standard Tet + Str + Cam Expresses seven rare tRNAs; acil itatesexpression o genes that encode rare E.

colicodons

Kan sensitive, trxB/gormutant, greatlyacilitates cytoplasmic disulde bond

ormation, Leu auxotroph

RosettaBlue

(DE3)

Origami 2

(K-12)


RosettaBlue

(DE3)pLysS

Origami 2

(K-12)


RosettaBlue

(DE3)pLacI

Origami 2

(K-12)


Rosetta-gami 2(DE3) Genotype:

D(ara-leu)7697DlacX74DphoA PvuII phoR araD139 ahpC galE galK rpsL

(DE3) F[lac+ lacIqpro] gor522::Tn10 trxBpRARE2 (CamR, StrR, TetR)

Rosetta-gami 2(DE3) pLysS Genotype:

D(ara-leu)7697DlacX74DphoA PvuII phoR araD139 ahpC galE galK rpsL

(DE3) F[lac+ lacIqpro] gor522::Tn10 trxBpLysSRARE2 (CamR, StrR, TetR)

Rosetta-gami B(DE3) Genotype:F ompT hsdSB(rB

mB) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxBpRARE

(CamR, StrR, TetR)

Rosetta-gami B(DE3) pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB

pLysSRARE (CamR, StrR, TetR)

RosettaBlue (DE3) Genotype: :

endA1 hsdR17 (rK12+) supE44 thi-1 recA1 gyrA96 relA1 lac (DE3)

F[proA+B+ mK12lacIqZDM15::Tn10]pRARE (CamR, TetR)

RosettaBlue (DE3)pLysS Genotype: :

endA1 hsdR17 (rK12+) supE44 thi-1 recA1 gyrA96 relA1 lac (DE3)

F[proA+B+ mK12lacIqZDM15::Tn10]pLysSRARE (CamR, TetR)

Ordering Inormation

Description Size Catalogue

No.

Rosetta-gami 2(DE3) Competent Cells 0 .4 mL 71351-3

1 mL 71351-4

Rosetta-gami 2(DE3) pLysSCompetent Cells

0.4 mL 71352-3

1 mL 71352-4Rosetta-gami 2(DE3) pLacICompetent Cells

0.4 mL 71353-3

1 mL 71353-4

Rosetta-gami B(DE3) Competent Cells 0 .4 mL 71136-3

1 mL 71136-4

Rosetta-gami B(DE3) pLysSCompetent Cells

0.4 mL 71137-3

1 mL 71137-4

Rosetta-gami B(DE3) pLacICompetent Cells

0.4 mL 71138-3

1 mL 71138-4

Description Size Catalogue

No.

RosettaBlue(DE3) Competent Cells 0.4 mL 71059-3

1 mL 71059-4

RosettaBlue(DE3) pLysS Competent Cells 0.4 mL 71034-3

1 mL 71034-4RosettaBlue(DE3) pLacI Competent Cells 0.4 mL 71060-3

Rosetta-gami 2 Competent Cells Set 1 set 71432-3

Rosetta-gami B Competent Cells Set 1 set 71177-3

RosettaBlue Competent Cells Set 1 set 71079-3
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18/44

8 Competent Cells

Adjust expression levels with TunerStrain Competent Cells

Tuner strains are lacZYdeletion mutants o BL21, which

enable adjustable levels o protein expression throughout

all cells in a culture. The lacpermease (lacY) mutation

allows uniorm entry o IPTG into all cells in the popula-

tion. Unlike lactose (or arabinose), IPTG is a gratuitous

inducer that can enter E. colicells independently rom

Targetprotein

75 100 1000300200150 M IPTG

Changing IPTG concentration aectsexpression levels in Tuner (DE3) cells.

A pET-21d() recombinant vector was trans-ormed into Tuner(DE3) cells, which weregrown to an OD600 o 0.6 and expression in-duced in replicate cultures with the indicatedconcentrations o IPTG. Ater a 4 h inductionperiod, total cell protein was analyzed by

SDS-PAGE and Coomassie blue staining.

Toxic Protein or Unstable Target PlasmidTuner strains are lacZYdeletion mutants o BL21 that allow control over

the level o protein expression throughout all cells in a culture. Protect your

cells using pLysS strains (in concert with pET, pCDF, pRSF, pACYDuet, or

pCOLADuet constructs) or pLacI strains (in concert with pETBlue and pTriEx

constructs), which enable tight control o basal expression. The HMS174 and

NovaBlue strains may stabilize certain target genes whose products may cause

the loss o the DE3 prophage.

permease pathways. This allows induction with IPTG to

occur in a true concentration-dependent ashion that is

exceptionally uniorm throughout the culture. By adjusting

the concentration o IPTG, expression can be regulated

rom very low expression levels up to the robust, ully

induced expression levels commonly associated with pET

vectors. Lower level expression may enhance the solubility

and activity o dicult target proteins. These strains are

also decient in the lon and ompTproteases.


(cu/g)

Packaging

Format


& Application

Tuner BL 21 > 2.0 x 106 Standard None BL21 lacZYdeletion mutant; allows precise

control with IPTGTuner(DE3) BL 21 > 2.0 x 106 Standard None

Tuner(DE3)pLysS BL 21 > 2.0 x 106 Standard Cam

Tuner(DE3)pLacI BL 21 > 2.0 x 106 Standard Cam

TunerGenotype:

F ompT hsdSB(rB mB

) gal dcm lacY1

Tuner (DE3)Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 (DE3)

Tuner (DE3)pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 (DE3) pLysS (CamR)

Tuner DE3)pLacI Genotype:

F ompT hsdSB(rB mB

) gal dcm lacY1 (DE3) pLacI (CamR)

Ordering Inormation


Tuner(DE3) Competent Cells 0.4 mL 70623-3

1 mL 70623-4

Tuner(DE3) pLysSCompetent Cells

0.4 mL 70624-3

1 mL 70624-4


Tuner(DE3) pLacI Competent Cells 0.4 mL 70625-3

1 mL 70625-4

Tuner Competent Cells Set 1 set 70726-3
http://www.emdmillipore.com/products/70623-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70623-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70624-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70624-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70625-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70625-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70726-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70726-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70625-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70625-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70624-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70624-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70623-4?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70623-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/70726-3?cid=BIOS-C-EPDF-4003-1212-SP


19/44


20/44

0 Competent Cells

B834 is the parental strain or BL21. These hosts are methionine auxotrophs and allow high specic activitylabeling o target proteins with 35S-methionine and selenomethionine or crystallography. This strain is also

decient in the lon and ompTproteases.

Protein Labeling With SelenomethionineWhen you are ready to do crystallographic studies, the B834 strain enables

selenomethionine labeling in a lon and ompT-defcient background.


(cu/g)

Packaging

Format


& Application

B834(DE3) B strain > 2.0 x 106 Standard none metauoxtroph, parent o BL21, control

nonexpression2 host

B834(DE3)pLysS B strain > 2.0 x 106 Standard Cam metauxotroph, parent o BL21, general

expression3 host, 35S-met labeling

B834(DE3) Genotype:

F ompT hsdSB(rB mB

) gal dcm met(DE3)

BLR(DE3)pLysS Genotype:

F ompT hsdSB(rB mB

) gal dcm met(DE3) pLysS(CamR)

Ordering Inormation


B834(DE3) Competent Cells 0.4 mL 69041-3

1 mL 69041-4

B834(DE3)pLysS Competent Cells 0.4 mL 69042-3

1 mL 69042-4

*The Resistance column in the tables reer to selectable resistant marker(s)

possessed by the strain in the absence o target plasmids. Appropriate

concentrations or selection are as ollows:

Kan: 15 g/mL kanamycin

Cam: 34 g/mL chloramphenicol

Tet : 12.5 g/mL tetracycline

Ri: 200 g/mL riampicin

Str: 50 g/mL streptomycin

Strains with the pLacI plasmid are appropriate hosts or pTriEx (1.14)

and pETBlue vectors only.

These strains carry a mutation in ribosomal protein (rpsL) conerring

resistance to streptomycin; thereore streptomycin is not necessary to

maintain strain genotype. I using pCDF vectors, spectinomycin must be

used or antibiotic selection because rpsL mutation coners streptomy-

cin resistance.

2. In this context, non-expression means that the strain does not contain

the gene or T7 RNA polymerase and thereore will not express rom

target genes under the control o a T7 promoter. These strains may be

suited or expression rom E. colipromoters such as lac, tac, trc, and trp,

or or inection by CE6 or pET expression.

3. Expression means that the strain is aDE3 lysogen, i.e., it carries the

gene or T7 RNA polymerase under lacUV5control. It is thereore suitedor expression rom T7 promoters.

4. High-stringency means that the strain carries pLysS, a pET-compatible

plasmid that produces T7 lysozyme, thereby reducing basal expression o

target genes.
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21/44

2

VectorsWhether you need to preserve and propagate a DNA sequence

o interest, or whether you need to express a recombinant

protein by delivering an engineered plasmid into bacteria,

EMD Millipore provides a large selection o cloning and

expression vectors, designed or your specifc needs. And

with our vectors and cloning kits, you can generate high

perormance constructs in as little as 40 minutes.

Il.


22/44


23/44


24/44

4 Vectors

Expression VectorsEfciently express your target protein by cloning your gene into one o our panel o

careully designed expression plasmids. Oering a choice o bacterial, mammalian or

insect cell systems, EMD Millipores vast portolio o expression vectors enables you to

choose the perect combination o promoters, epitope tags, antibiotic resistance, andhost compatibility.

Vector Family Host System(s) Product Highlights

Gateway Nova pET-DEST

& pCOLA-DEST Vectors

Bacterial Reliable pET expression & purication with rapid Gateway cloning

Duet Vectors Bacterial T7 promoter expression vectors

Coexpression o multiple target proteins

LIC Duet Adaptors Bacterial Clone two open reading rames at once into E. coliEk/LIC vectors or coexpression

pBAC Vectors Insect Baculovirus transer plasmids designed or convenient cloning and expression o

target genes rom baculovirus vectors

pBiEx Vectors Insect/Bacterial Rapid expression o target genes in both E. coliand insect cells

pCDF and pRSF Vectors Bacterial Designed with compatible replicons and drug resistance or co-expression with each

other or with many other pET vectors

pET Vectors Bacterial Most requently cited system or prokaryotic protein expression

Highest expression levels, tightest control over basal expression

pETBlue Vectors Bacterial Tightly controlled T7-driven expression plus blue/white screen and high plasmid yield

pETcoco Vectors Bacterial Controllable-copy number vectors or improved stability o constructs

plEx Vectors Insect Rapid, high-level protein expression in insect cells without creating a recombinant

baculovirus

pIEx/Bac Vectors Insect Dual-purpose vectors or direct transection into insect cells or baculovirus

production

pT7Blue Vectors Bacterial Archiving, subcloning, sequencing, in vitrotranscription

pT7Blue-2 is also suitable in vitrotranscription/translation & sequencing

pSCREEN Vectors Bacterial The pSCREEN T-Vector is designed or expression o inserts as stable usion proteinsdriven by T7 RNA polymerase.

pSTBlue Vectors Bacterial Multi-purpose cloning vector eaturing a versatile multiple cloning region, blue/white

screening, dual opposed T7/SP6 promoters and dual Kan/Amp resistance

pTandem-1 Vector and pTK-neo Mammalian pTandem vector is designed or coexpression o two genes in mammalian cells rom

a bicistronic RNA

pTK-neo vector can select stably transormed mammalian cell lines using G418

pTriEx Vectors Bacterial/insect/

mammalian

Enables optimal protein expression in bacterial, insect and mammalian cells rom a

single plasmid

T7Select Vectors Bacterial Phage display vectors

For more inormation, please reer to the Vector Selection Tool at www.emdmillipore.com/vectors
http://www.emdmillipore.com/life-science-research/novagen-vector-tables/c_d0ub.s1OqxsAAAEhv.4vVcUP?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/life-science-research/novagen-vector-tables/c_d0ub.s1OqxsAAAEhv.4vVcUP?cid=BIOS-C-EPDF-4003-1212-SP


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26/44


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28/44

8 Vectors

Ordering Inormation


Gateway Nova pET-53-DEST Expression System 1 Kit 71856-3

Gateway Nova pET-54-DEST DNA 10 g 71845-3



Gateway Nova pET-57-DEST Expression System 1 Kit 71860-3






Gateway Nova pCOLA-3-DEST DNA 10 g 71854-3

Ligation-Independent Cloning (LIC) Vectors

Ligation-Independent Cloning (LIC) Vector Kits oer rapid, efcientdirectional cloning o PCR products, without restriction enzyme digestion

or ligation reactions. The LIC method provides an alternative an alternative

to recombinase-mediated cloning with the unique advantage that, ater

purifcation, all N-terminal vector-encoded sequences can be removed.

Three amilies o LIC vectors are available: Ek/LIC (enterokinase), Xa/LIC

(Factor Xa) , and 3C/LIC (HRV 3C).

Plasmid replicons in EMD MilliporeE. coliexpression systems

Plasmid(s) Replicon (source) Replicon (source)

pET (all)

pETDuet-1

ColE1 (pBR322) ~40*

pET-DEST ColE1 (pBR322) ~40

pACYCDuet-1

pLysS

pLysE

pLacI

pRARE

P15A (pACYC184) 1012

pRSF (all) RSF1030 > 100

pCDF (all) CloDF13 20-40

pETBlue (all)

pTriEx (all)

ColE1 (pUC) > 500

pETcoco (all) Mini-F/RK2 (2)

(pBeloBAC11, RK2)

1, ampliable

to ~40

pCOLA-DEST ColA ~20-40

pCOLADuet-1 ColA ~20-40

* Copy number estimates are based on agarose gel analysis

Amplify target usingany thermostableDNA polymeraseand special primers

ANNEAL

5 min

TRANSFORM

~ 1 hour

PLATE

LIC Vector

After 5 min., add EDTA

PCR productT4 DNA polymerasedATO ir dGTP

NovaBlue GigaSingles

Competent Cells

SOC Medium

Procedure time:

30-minute polymerase treatment

20-minute heat inactivation

5-minute annealing

8-minute transormation

60-minute outgrowth
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30/44


31/44

3Vectors

Insect Expression SystemsUsing insect cells or recombinant protein expression is not a new process.

However, this process has traditionally been tedious and labor intensive.

EMD Millipores insect cell expression systems have harnessed the recent

advancements in insect cell-mediated expression, making this a useul tool

or recombinant protein expression.

The original BacMagic System allows you to generate

recombinant baculoviruses without the time-consuming

plaque purication process. The next generation o the

BacMagic System has the added advantage o improved

quality and yield or most target proteins, through thedeletion o additional non-essential genes. BacMagic-2

DNA deletes chiA and v-cath genes, resulting in signi-

cantly improved quality and yield or most target proteins.

The BacMagic-3 DNA combines the deletion ochiA

and v-cath with the additional deletion o three more

nonessential virus genes, p10, p74 and p26, or more

ecient expression.

Faster baculovirus production without tediousplaque purication

Compatible with pIEx/Bac, pBAC, pTriEx, and othertranser plasmids using the lef2/603 and ORF1629

recombination sites

Deletion o chitinase to maximize secreted andmembrane targeted protein production

Includes Insect GeneJuice Transection Reagent orhigh eciency transection

BacMagic Systems

Ordering Inormation


BacMagic-3 Transection Kit 5 reactions 72351-3

BacMagic-3 DNA Kit 5 reactions 72350-3

Not available in Japan

BacMagic 3 System: Improved quality and yield

with no plaque purifcation needed!

DAY 1 DAY 2 DAY 3 DAY 4

Cotransect insect

cells with recombinant

transer plasmid plus an

AcNPV BacMagic DNA

Harvest recombinant

baculovirus; screen or

expression; ampliy

viral stock (titer stock,

optional)

Inect insect cells and

express protein

Harvest cells and proceed

with purication

chiA

GeneORF1

629

Recombinant virus

BacMagic-3 DNA

chiA

lef2 Bac

Gene ORF1629

lef2

lef2

1629

Transfer plasmid

v-cath

v-cath

p10

p10

Homologous recombination schemes o

the BacMagic-3 System
http://www.emdmillipore.com/products/72351-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/72350-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/72350-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/72351-3?cid=BIOS-C-EPDF-4003-1212-SP


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2 Vectors

pIEx/Bac Expression Vectors

pIEx/Bac dual purpose vectors, together with BacMagic System, provide

the perect combination or baculovirus inection and expression. Because the

pIEx/Bac vectors are compatible with both plasmid-mediated and baculovirus

expression, it is easy to use a single construct in both expression systems orhigh throughput screening and optimized high yield protein expression.

BacMagic Systems

AcNPV hr5/ie1 promoter/enhancer or plasmid

mediated and early baculovirus expression

AcNPVp10very late promoter or late

baculovirus expression

N-terminal and C-terminal usion tags or dual

purication strategies

Available as LIC Vector Kits or ecient, fexible

ligation-independent cloningpurication strategies

10

15

25

35

50

75

100

150

M 1 2 3 4 5 6 7 8 M

225

Insect cell expression using pIEx/Bac-4 and 5 dual-purpose vectors.

Open reading rames or two proteins (CHK1or Rluc) were cloned into pIEx/Bac-4 and5 vectors. Plasmid-mediated and baculo-virus-mediated expression was analyzed oreach protein in each vector. Expressed targetproteinswerepuriedusingGSTBindResinin batch mode. The eluted proteins wereassayed by SDS-PAGE and visualized withRAPIDstain Reagent.

Ordering Inormation


pIEx/Bac-1 Ek/LIC Vector Kit 20 reactions 71729-3

pIEx/Bac-3 3C/LIC Vector Kit 20 reactions 71731-3

pIEx/Bac-4 Ek/LIC Vector Kit 20 reactions 71732-3

pIEx/Bac-5 3C/LIC Vector Kit 20 reactions 71733-3

Lane Sample

M Perect Protein Markers, 10-225 kDa

1 Inection, pIEx/Bac-4, CHK1 (81.6 kDa)

2 Transection, pIEx/Bac-4, CHK1 (81.6 kDa)

3 Inection, pIEx/Bac-5, CHK1 (83.9 kDa)

4 Transection, pIEx/Bac-5, CHK1 (83.9 kDa)

5 Inection, pIEx/Bac-4, Rluc (64.3 kDa)

6 Transection, pIEx/Bac-4, Rluc (64.3 kDa)

7 Inection, pIEx/Bac-5, Rluc (65.5 kDa)

8 Transection, pIEx/Bac-5, Rluc (65.5 kDa)

M Perect Protein Markers, 10-225 kDa
http://www.emdmillipore.com/products/71729-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71731-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71732-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71733-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71733-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71732-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71731-3?cid=BIOS-C-EPDF-4003-1212-SPhttp://www.emdmillipore.com/products/71729-3?cid=BIOS-C-EPDF-4003-1212-SP


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4

Culture MediaMedia are easy to take or granted. A staple o every protein

expression laboratory, media fll countless rows o asks and

test tubes. Untold hours o media mixing and autoclave run

time should not be the bottleneck to obtaining experimental

data. On the other hand, reliable media are critical or reliable

experimental results. We oer a wide range oE. colimedia in

ormats that reduce media preparation time. Spend less time

measuring (and inhaling) powdered mediaEasyPak media

packets are ready to go just add water and sterilize*.

Your E. colicultures depend on consistent, high-quality media

and happy cells make better protein.

Ill.


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6 Culture Media

Overnight Express Protocol Conventional Protocol

Overnight Express Autoinduction SystemsWith Overnight Express Autoinduction Systems, a period o cell growth is

ollowed by spontaneous induction o protein expression without monitoring

cell density and without conventional induction with IPTG. The method is based

on media components that are metabolized dierentially to promote growth to

high density and automatically induce protein expression rom lacpromoters.

Novagen

OvernightE

xpress

InstantTB

Medium

Prepare medium by adding

1 EasyPak of medium into

1 liter of water

INOCULATE

INCUBATE

HARVEST

HARVEST

Weigh out

various ingredients

Repeat untilculture is readyto be induced.

PREPARE MEDIUM

INOCULATE

INCUBATE

INCUBATE

CHECK on OD

ADD INDUCER


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4

AntibioticsAntibiotics are common selection agents or bacteria

carrying selectable markers. A selectable marker, oten a

bacterial resistance gene, is a gene which coners a trait

suitable or artifcial selection. Bacteria that have been

transormed with oreign DNA are grown on a medium

containing an antibiotic, and those bacterial colonies that can

grow have successully taken up the DNA. In most applications,

only one in a several billion cells will take up DNA. Rather than

checking every single cell, use a selective antibiotic to kill all

cells that do not contain the oreign DNA, leaving only the

desired ones. Choose rom our wide variety o antibacterial

antibiotics to maintain the bacterial genotypes o your choice.

V.


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2 Antibiotics

CarbenicillinCarbenicillin intereres with bacterial cell wall synthesis.

It is recommended or use in place o ampicillin to

maintain the selective marker bla(b- lactamase, which

coners resistance to ampicillin). Ampicillin is degraded

by endongenous secreted b-lactamase enzyme and by

the drop in pH that usually accompanies bacterial

ermentation. Carbenicillin is more stable at low pH

than ampicillin, preventing loss o drug resistance.

ChloramphenicolChloramphenicol is a synthetic bacteriostatic antibiotic

that inhibits translation o RNA by blocking the

peptidyltranserase reaction o ribosomes.

G 418 SulateG 418 Sulate is an aminoglycoside that inhibits prokary-

otic and eukaryotic protein synthesis. IT is widely used in

the selection o eukaryotic vectors carrying neomycin or

kanamycin resistance genes. The product o these genes,

aminoglycoside 3-phosphotranserase, inactivates G 418,

neomycin and kanamycin by phosphorylation.

Hygromycin BStreptomycessp.

Hygromycin is an aminoglycoside antibiotic that inhibits

growth o prokaryotic (bacteria) and eukaryotic (yeast)

microorganisms and mammalian cells It inhibits protein

synthesis at the translocation step on the 70S ribosome

and causes mRNA misreading. Hygromycin B penetrates

cells that have been permeabilized by virus inection;

hence, it can act as an eective antiviral agent.

Kanamycin Sulate,Streptomyces kanamyceticussp.

Kanamycin Sulate is an aminoglycoside antibiotic

eective against Gram-positive and Gram-negative

bacteria. It inhibits protein biosynthesis by acting on

the 30S ribosome, causing misreading o the genetic code.

In mammals, kanamycin may cause renal damage and

is ototoxic.

Spectinomycin,Dihydrochloride,PentahydrateStreptomycessp.

Spectinomycin is a broad-spectrum aminoglycoside

antibiotic containing two glucose moieties. It is eective

against Gram-positive and Gram-negative bacteria. It

inhibits initiation, elongation, and termination o protein

synthesis in prokaryotes and induces misreading o the

genetic code. Footprint studies indicate that spectinomycin

exerts regional eects on ribosomal structure.

Streptomycin SulateStreptomycessp.

Streptomycin Sulate is an antibiotic eective against

Gram-positive and Gram-negative bacteria. It inhibits

initiation, elongation, and termination o protein synthesis

in prokaryotes, induces misreading o the genetic code.

It is oten used in culture media to control growth o

microorganisms.

Puromycin, DihydrochlorideAn aminonucleoside antibiotic that acts as a prokaryotic

and eukaryotic protein synthesis inhibitor. Resembles the

aminoacyl-adenylyl terminus o aminoacyl-tRNA andcompetes or binding to the A-site o the large ribosomal

subunit.

Tetracycline, HydrochlorideTetracycline, Hydrochloride blocks protein synthesis

by inhibiting aminoacyl tRNA binding to the A-site o

ribosomes. It induces a cold shock-response and enhances

P450 expression in bacteria.


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Documents

Competent cells, vectors, media & antibiotics for recombinant protein production Product Guide