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/

[math]\displaystyle{ \lambda }[/math] phage exo, bet, and gam genes are integrated into E-coli to demonstrate phage promoted homologous recombination. Murphy 1998 [math]\displaystyle{ \lambda }[/math] Red-Promoted Gene Replacement

To acheive control over phage recombination gene activity, temperature sensitive [math]\displaystyle{ \lambda }[/math] 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. 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 [math]\displaystyle{ \lambda }[/math] phage recombination proteins, exonuclease, [math]\displaystyle{ \beta }[/math], and [math]\displaystyle{ \gamma }[/math]!!

An Exonuclease Induced by phage [math]\displaystyle{ \lambda }[/math]. Little 1967

Roles of Exonuclease and [math]\displaystyle{ \beta }[/math] protein of phage [math]\displaystyle{ \lambda }[/math] in recombination. Carter 1971

[math]\displaystyle{ \beta }[/math] protein of phage [math]\displaystyle{ \lambda }[/math] promotes renaturation of DNA. Kmiec 1981

Pairing activities of [math]\displaystyle{ \beta }[/math] protein in phage [math]\displaystyle{ \lambda }[/math]. Muniyappa 1986

How [math]\displaystyle{ \gamma }[/math] protein specified by phage [math]\displaystyle{ \lambda }[/math] affects recBC enzyme in E. Coli. Karu 1975

[math]\displaystyle{ \gamma }[/math] protein of phage [math]\displaystyle{ \lambda }[/math] inhibits recombination activites of RecBCD enzyme. Murphy 1991