Recombineering
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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/
λ phage exo, bet, and gam genes are integrated into E-coli to demonstrate phage promoted homologous recombination.
Murphy 1998 λ Red-Promoted Gene Replacement
To acheive control over phage recombination gene activity, temperature sensitive λ 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 λ phage recombination proteins, exonuclease, β, and γ!!
An Exonuclease Induced by phage λ. Little 1967
Roles of Exonuclease and β protein of phage λ in recombination. Carter 1971
β protein of phage λ promotes renaturation of DNA. Kmiec 1981
Pairing activities of β protein in phage λ. Muniyappa 1986
How γ protein specified by phage λ affects recBC enzyme in E. Coli. Karu 1975
γ protein of phage λ inhibits recombination activites of RecBCD enzyme. Murphy 1991
