Easy Gene Splicer DNA Ligation & Colony Transformation

Carolina KitIsabelle Muschamp

1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme)

2. DNA ligase joins the DNA fragment & vector DNA

3. Host cell is made competent so can plasmid can enter

4. Transformed cells are grown on selection media

DNA Ligation & Colony Transformation Overview

Extrachromosomal DNA molecules Small; varies from 1 to over 1,000 kilobase pairs Circular Usually carry one or a few genes Replicate separately from the chromosome A type of cloning vector used to carry a gene not found in the bacterial host’s chromosome Treatment of plasmids pAMP and pKAN with a mixture of BamHI

and HindIII are given for the Easy Gene Splicer lab

Plasmids

4539 base pairs a single replication origin a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of

penicillin) a single occurrence of the sequence 5' GGATCC 3'

3' CCTAGG 5‘ that, as we saw above, is cut by the restriction enzyme BamHI a single occurrence of the sequence 5' AAGCTT 3'

3' TTCGAA 5‘ that is cut by the restriction enzyme HindIII Treatment of pAMP with a mixture of BamHI and HindIII produces: -a fragment of 3755 base pairs carrying both the ampr gene and the

replication origin -a fragment of 784 base pairs -both fragments have sticky ends

pAMP

4207 base pairs a single replication origin a gene (kanr) conferring resistance to the antibiotic

kanamycin a single site cut by BamHI a single site cut by HindIII Treatment of pKAN with a mixture of BamHI and

HindIII produces: -a fragment of 2332 base pairs -a fragment of 1875 base pairs with the kanr gene

(but no origin of replication) -both fragments have sticky ends

pKAN

Both the DNA that will be inserted and the plasmid DNA are ligated together

Sticky ends help this process by stabilizing the pieces together with base-pairing, but the enzyme DNA ligase is added to the reaction to cause the DNAs to become covalently linked together

DNA Ligation

After ligation of the insert DNA and your plasmid, the new chimeric (an organism that is composed of genetically different tissues, either naturally or as a result of a laboratory procedure) of DNA into bacteria

Transformation of the bacteria with recombinant DNA is easily performed by shocking the cells

Colony Transformation

The process of transferring foreign DNA fragments into a recipient (host) cell for growth and replication

Scientists begin this process by fusing two different sets of DNA together creating a molecule of recombinant DNA or rDNA. This particular molecule is the product of the gene of interest (the desired gene) and another source of plasmid DNA coming together through the use of a restriction enzyme, which cuts the DNA at a specific nucleotide sequence, and a ligase which binds the separate strands of DNA into one form.

After the rDNA is made, the cells which are going to be used as a vehicle for transformation are made “competent” or prepared to receive foreign DNA.

DNA Ligation & Colony Transformation

The cells are concentrated into a pellet through the use of a centrifuge, and their membranes are made porous so that the rDNA has a route to enter the cell.

The rDNA is added to the cell culture and some of the rDNA plasmids are absorbed, but to increase their absorption numbers the culture undergoes a heat/cold shock. The hot water bath enlarges the cell’s pores and more plasmids are “sucked” in. The culture is then quickly transferred to the ice which traps the plasmids within the cell’s membrane.

Cells containing the foreign DNA grow and multiply within the tube, but to ensure that transformation was successful and purification of the gene of interest to proceed, the culture is grown on mediums so visual confirmation can be made.

DNA Ligation & Colony Transformation Continued

Endonucleases: ◦ in nature, they protect bacteria from intruding DNA◦ cut up (restrict) the viral DNA◦ cut only at very specific nucleotide sequences

Restriction site:recognition sequence for a particular restriction enzyme

Restriction fragments:segments of DNA cut byrestriction enzymes in a reproducible way

DNA ligase: joins the sticky ends of DNA fragments

Restriction Enzymes

rDNA (recombinant DNA)—the produced piece of DNA from inserted another piece of DNA Recognize specific sites to cut the DNA Blunt ends—straight across Sticky ends—one side of DNA is longer than the other, these overhangs allow for complementary matches between two DNA pieces cut by the same enzyme, the sticky ends match and pasting may occur to produce an rDNA molecule More than 1200 restriction enzymes discovered & isolated from bacteria Read 5 3 Palindromic (example radar or

GAATTCCTTAAG

Restriction Enzymes Continued

• Transformation – the uptake and expression of foreign DNA by a cell• Transduction – the use of viruses to transform or genetically engineer cells• Competent/competency – the ability of cells to take up DNA• Selection – the process of screening potential clones for the expression of a

particular gene, for example, the expression of a resistance gene (such as resistance to ampicillin) in transformed cells

• Transformation efficiency – a measure of how well cells are transformed to a new phenotype

• Recovery period – the period following transformation where cells are given nutrients and allowed to repair their membranes and express the “selection gene(s)”

• Beta-galactosidase gene – a gene that produces beta-galactosidase, an enzyme that converts the carbohydrate X-gal into a blue product

• Green fluorescent protein – a protein found in certain species of jellyfish that glows green when excited by certain wavelengths of light (fluorescence)

• Scale-up – the process of increasing the size or volume of the production of a particular product

Restriction Enzyme Vocabulary

Transformation Applications

Week Before: read lab & Flowchart, only 6 groups

Thursday—Lecture, pre-quiz Monday—part A Tuesday—part B Thursday--part C Friday FIRE—come view results

Timeline

Prepare • LB agar plates (<1 month before)

2 LB agar plates for streaking to use in part B16 (gives 4 extra) LB agar plates for part C

12 (gives 4 extra) LB agar plates with ampicillin and kanamycincoming with kit—check for 2 LB/amp and 2 LB/kan for control group

• Aliquot for 6 groupsPart A--20uL Ligation Buffer/ATP/ligase (6)--10uL pAMP (5 tubes—pKAN control does not get one)--10uL pKAN (5 tubes—pAMP control does not get one)Part B--500uL Calcium Chloride (6)--500uL LB (6)

Locate Supplies: Spreaders loopsset up for part A iceset up for part B Streak starter plates (12-20 hours before part B) (start after school) 42C water bath 37C incubator put calcium chloride on ice When finish store at 0C (beaker of ice in fridge)set up for part C 37C incubator

Preparation For Lab

2 lab station groups will be control groups: 1. -–pAMP + ligase (use 10uL water)

2. --pKAN + ligase (use 10uL water)

Follow same directions, but run these instead of pAMP/KAN

Only plate on LB/amp and LB/kan plate (do not spread on LB plates)

Controls

Group 1—pAMP control

Group 2—pKAN control

Group 3

Group 4

Group 5

Group 6

Group Assignments

http://bioinformatics.dnalc.org/gmo/animation/gmo.html

http://www.carolina.com/text/teacherresources/instructions/biotech/ez_gene_splicer_kit.pdf

Resources

• Endonucleases – enzymes that cut RNA or DNA at specific sites; restriction enzymes are endonucleases that cut DNA

• Sticky cells – restriction fragments in which one end of the double stranded DNA is longer than the other; necessary for the formation of recombinant DNA

• Restriction enzyme mapping – determining the order of restriction sites of enzymes in relation to each other

Terminology

Samples of the plasmid fragments are mixed with DNA ligase and incubated at room temperature for 2-24 hours. Complementary BamHI and HindIII “sticky ends” hydrogen-bond to align restriction fragments. Ligase catalyzes the formation of phosphodiester bonds that covalently link the DNA fragments to form stable recombinant DNA molecules.

Use micropipets (instead of transfer) After 2-24hours, will store in fridge at 4C

Part A

Transform E.coli with the ligated plasmid DNA. E. coli cells are scraped off an LB agar plate and suspended in two tubes containing solution of calcium chloride. The ligated pAMP/KAN plasmid is added to one cell suspension, and both tubes are incubated at 0C, then heat shocked at 42C, cooling and addition of LB broth, cells then recover.

I will store cells at 0C (beaker of ice in fridge) after 5-6 hours of incubation

Part B

Samples of the cells are plated on two types of growth media: plain LB agar and LB agar with ampicillin and kanamycin. Incubate to grow cells. Only cells with both ampicillin and kanaycin will be expressed on the 2nd plate

Come in at FIRE to see You are receiving 2 LB plates—so you can

do both plates in PART C step 1.C and 2 Use spreaders at Part C 3

Part C