Targeted Gene Knockout and Essentiality Testing by Homologous Recombination
This chapter provides an updated experimental protocol for generating allelic exchange mutants of mycobacteria by two-step selection using the p2NIL/pGOAL system. The types of mutants that can be generated using this approach are targeted gene knockouts marked with a drug resistance gene, unmarked deletion mutants, or strains in which a point mutation/s has been introduced into the target gene. A method for assessing the essentiality of a gene for mycobacterial growth by means of allelic exchange is also described. This method, which utilizes a merodiploid strain carrying a second copy of the gene of interest on an integration vector, allows the exploration by means of complement switching of structure–function relationships in proteins that are essential for mycobacterial growth.
Key wordsAllelic exchange Complement switching Homologous recombination Illegitimate recombination Phthiocerol dimycocerosate (PDIM) UV irradiation
This work was financially supported by grants from South African Medical Research Council (to V.M.), the National Research Foundation (to V.M.), and the Howard Hughes Medical Institute (Senior International Research Scholar’s grant to V.M.).
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