title: Targeted gene knockout and essentiality testing by homologous recombination.
abstract: 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.
authors.affiliation: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
authors.forename: Krishnamoorthy
authors.initials: K
authors.lastname: Gopinath
authors.forename: Digby F
authors.initials: DF
authors.lastname: Warner
authors.forename: Valerie
authors.initials: V
authors.lastname: Mizrahi
mesh_terms: D000483:Alleles; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D041722:Genes, Transgenic, Suicide; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics