λred recombineering Flashcards
In homologous recombination, what protein forms a filament with ssDNA?
RecA
What is λred?
A bacteriophage that encodes a system that promotes homologous recombination.
What is λred Exo?
A 5’-3’ exonuclease that degrades linear DNA from the 5’ end
What is λred Beta?
A protein that binds to the ss 3’ ends generated by Exo. It promotes annealing to comp. DNA
What is λred Gam?
An inhibitory protein that binds to the host RecBCD complex, halting host exonuclease activity.
How long are the homologous sequences in this system?
30-50bp
pKD46 contains which genes?
The λred genes.
How is pKD46 activated?
Arabinose induction.
What genetic elements are on the linear DNA used in conjunction with pKD46?
a1/a2 regions, r1/2 restriction sites and the replacement gene.
Why should λred genes be removed from the host?
They can promote unwanted events.
What does pKD46 feature to allow λred gene removal?
A temperature sensitive replicon.
Which two plasmids are used in the Gene Doctoring technique?
pACBSCE & pDOC
In Gene Doctoring, what counter-selection gene is used?
sacB
How does sacB counter-selection work?
The sacB product kills gram-ve bacteria when sucrose is added.
pACBSCE features genes for SceI. What is SceI.
A meganuclease that cuts specific sites.
