Recombineering

Protocols

 * λ red-mediated gene replacement — Datsenko and Wanner method, using plasmids
 * Court lab λRed temperature sensitive system — Court method using temperature sensitive plasmids (pSIM5)

Resources
Recombineering is a method where E-coli undergoes recombination between linear DNA, introduced through electroporation, with circularized DNA already present within the cell.

http://recombineering.ncifcrf.gov/

$$\lambda$$ phage exo, bet, and gam genes are integrated into E-coli to demonstrate phage promoted homologous recombination. Murphy 1998 $\lambda$ Red-Promoted Gene Replacement

To acheive control over phage recombination gene activity, temperature sensitive $$\lambda$$ repressor (cI857) was introduced. Yu 2000 An efficient recombination system for chromosome engineering in Escherichia coli

Court Lab.

This process is then adapted to E-coli strain DH10B, a Bacteria Artificial Chromosome (BAC)strain. This results in the original recombineering E-coli strain designated DY380 as well as other versions modified to include different selection mechanisms. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11352566 Lee 2001. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA]

Church Lab.

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization-

All about the $$\lambda$$ phage recombination proteins, exonuclease, $$\beta$$, and $$\gamma$$!!

An Exonuclease Induced by phage $$\lambda$$. Little 1967

Roles of Exonuclease and $$\beta$$ protein of phage $$\lambda$$ in recombination. Carter 1971

$$\beta$$ protein of phage $$\lambda$$ promotes renaturation of DNA. Kmiec 1981

Pairing activities of $$\beta$$ protein in phage $$\lambda$$. Muniyappa 1986

How $$\gamma$$ protein specified by phage $$\lambda$$ affects recBC enzyme in E. Coli. Karu 1975

$$\gamma$$ protein of phage $$\lambda$$ inhibits recombination activites of RecBCD enzyme. Murphy 1991