Our group continues to develop innovative methods for the high-throughput manipulation of the mycobacterial chromosome.
Current methodology is centered on a "drag and drop" engineering approach called ORBIT (“oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting”). A synthetic "targeting oligonucleotide" is integrated into the chromosome via a RecT-like annealase, creating an recombination site for the simultaneous integration of a circular "payload plasmid". Any number of functionally-distinct mutations can be generated by combining a single targeting oligo with existing payload plasmids.(Murphy KC, Nelson SJ, Nambi S, Papavinasasundaram K, Baer CE, Sassetti CM. 2018. ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes. mBio 9:e01467-18. https://doi.org/10.1128/ mBio.01467-18.)
Payload plasmids are available from Addgene. We encourage others that produce plasmids with new functionalities to add them to this library: