Abstract
The precise knockout or modification of Mycobacterium tuberculosis genes has been critical for the identification of functions important for the growth and pathogenicity of this important bacterium. Schemes have been previously described, using both non-replicating vectors and transducing particles, for the introduction of gene knockout substrates into M. tuberculosis, where the endogenous recombination systems of the host (both homologous and illegitimate) compete for transfer of the modified allele to the chromosome. Recombineering technologies, first introduced in laboratory and pathogenic strains of Escherichia coli over the last 16 years, have been developed for use in M. tuberculosis. Described in this chapter is the use of the mycobacterial Che9c phage RecET recombination system, which has been used to make gene knockouts, reporter fusions, promoter replacements, and single base pair modifications within the M. tuberculosis and M. smegmatis chromosomes at very high frequency. Higher success rates, in a shorter period of time, are routinely observed when recombineering is compared to previously described M. tuberculosis gene knockout protocols.
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Acknowledgements
We would like to acknowledge Graham Hatfull and Julia van Kessell for providing the che9c recombination genes and invaluable advice, Adrie J.C. Steyn for the pCreSacB-Kan plasmid, Dirk Schnappinger for reagents and advice, and Chitra Kanchagar for expert technical assistance. This work was supported by the Bill and Melinda Gates Foundation, the NIH (AI0645282, AI095208, and U19AI107774), and the Howard Hughes Medical Institute.
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Murphy, K.C., Papavinasasundaram, K., Sassetti, C.M. (2015). Mycobacterial Recombineering. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_10
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DOI: https://doi.org/10.1007/978-1-4939-2450-9_10
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