Pavelka MS Jr (2008) Allelic exchange of unmarked mutations in Mycobacterium tuberculosis. Key wordsĪzad AK, Sirakova TD, Rogers LM et al (1996) Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides. 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. 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. 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. 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. ![]() In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens.
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