The lambda red recombination system is used to selectively induce homologous recombination.
The three protein-coding genes required for this system are delivered by a plasmid delivery system, containing gam, beta, and exo. The gene(s) of interest being recombined into the host bacteria is delivered by transformation (normally by electroporation), with flanking oligonucleotide primers containing the regions of homology.
Exo converts dsDNA to ssDNA, which is able to be inserted at the homologous regions (which can be just 40 base pairs long with lambda red).
Beta binds to the ssDNA 3’ end, promoting the homologous region annealing with the chromosomal DNA.
Gam binds to the host’s RecBCD complex, inhibiting exonuclease activity and allowing the recombination event to occur successfully.
These three genes are transformed to the target bacteria on the pKD46 plasmid. This is a temperature sensitive plasmid, degrading at higher growth temperatures. While recombineering is being carried out, the bacteria are grown at the permissive temperature of 30C, before being transferred to a higher temperature to catalyse the plasmid’s degradation. As the lambda red genes are under an araC promoter, they are only expressed while the bacteria are being grown on arabinose-containing media.
Once the transformation of pKD46 has been completed, and sufficient time allowed for the recombination before removing pKD46 (by growing under the restrictive conditions). A selectable marker, such as antibiotic resistance, can be selected for to identify recombinants. CRISPR-Cas can also be used to identify successful recombinants. By also deleting a PAM site during the recombineering process, it is possible to induce ds breaks in bacteria that have not undergone homologous recombination successfully. As bacteria have less robust DNA repair mechanisms than in eukaryotic cells, this screening technique will result in the death of any cells not containing the desired genes.