Oligo-Mediated Recombineering and its Use for Making SNPs, Knockouts, Insertions, and Fusions in Mycobacterium...

Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This "recombineering" technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to) 500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events, including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.