MBB 267 Week 5: Corrigan 3

Question 1

What is homologous recombination used for?

Answer 1

used to create specific gene replacements/knockouts in bacteria
a. The most commonly used system is lambda Red

Question 2

How does homologous recombination occur?

Answer 2

So

a. RecBCD enters end of DNA fragment and unwinds it. When it reaches a Chi site (8 bp sequence) it nicks the DNA and continues to unwind the DNA.
b. A RecA filament assembles on the ssDNA.
c. RecA scans the dsDNA for homology.
d. RecA catalyses strand invasion and D-loop formation (single-strand crossover).
e. RuvAB assemble at the crossover point and pull the donor and recipient strands in opposite directions (branch migration).
f. Endonuclease cleaves one end of the D- loop.
g. Displaced ends are ligated to opposite strands.
h. Holliday structure is resolved by RuvC cleaving the DNA across the junction.
i. Ligation of broken ends completes a single crossover.

Question 3

What is a cointegrate form?

Answer 3

A cointegrate is the intermediate molecule between donor DNA and target DNA covalently bind during the formation of a Holliday junction.

Question 4

What are the differences that arise if the donor and recipient DNAs were circular or linear?

Answer 4

2 types;

a. If donor and recipient were circular, then a cointegrate forms
b. If the donor was linear and the recipient circular, then a second crossover is needed to maintain viability (circularity). The recipient now contains donor DNA.

Question 5

How do gene knockouts work in Gram Positive bacteria?

Answer 5

Need:

a. A PCR product with at least 1 kb of homologous regions on both sides of the gene.
b. Then homologous recombination works by Rec enzymes.

Question 6

what is branch migration?

Answer 6

Branch migration is the process by which base pairs on homologous DNA strands are consecutively exchanged at a Holliday junction, moving the branch point up or down the DNA sequence.

Question 7

what is lambda Red?

Answer 7

Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering. The lambda red system is an alternative method that can be used for cloning or genome engineering and is based on homologous recombination. It allows for direct modification of DNA within E. coli and is independent of restriction sites.

Question 8

What does bacteriophage lambda do?

Answer 8

The bacteriophage lambda encodes a system (Red) that promotes homologous recombination

Question 9

What does system red use for proteins?

Answer 9

Genes are carried on a plasmid;

a. Genes that are switched on by arabinose induction.
i. Exo: 5’-3’ exonuclease that degrades 5’ ends of linear DNA (in this system a PCR product)
ii. Beta: binds to the single stranded 3’ ends generated by Exo and promotes annealing to complementary DNA (bacterial chromosome)
iii. Gam: Gam binds to the host RecBCD complex to inhibit exonuclease activity
b. Other genes include;
i. Bla: antibiotic resistance cassette
ii. oriR101: origin site of replication
iii. repA101ts: temperature sense at 42 degrees

Question 10

Dangers of red genes?

Answer 10

Could promote unwanted events

Question 11

When should red genes be removed?

Answer 11

Remove as soon as desired construct is obtained

Question 12

How do we remove red genes?

Answer 12

pKD46 has a temperature sensitive replicon – culture at 42°C

Question 13

How does the new doctoring gene work?

Answer 13

Itroduce 2 plasmids; pACBSCE and pDOC

a. pACBSCE has 1-secI, which is an endonuclease which cleaves both plasmids and is induced by arabinose
b. Lambda red proteins facilitate the recombination between recipient DNA and linear DNA obtained by cutting pDOC plasmid with 1-secI
c. All other linear fragments are digested
d. Cells that have lost sacB gene and retained the kanamycin resistance cassette successfully can grow and survive on media supplemented with kanamycin and sucrose.