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Targeted Gene Knockout and Essentiality Testing by Homologous Recombination

  • Krishnamoorthy Gopinath
  • Digby F. Warner
  • Valerie MizrahiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)

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.

Key words

Allelic exchange Complement switching Homologous recombination Illegitimate recombination Phthiocerol dimycocerosate (PDIM) UV irradiation 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Krishnamoorthy Gopinath
    • 1
  • Digby F. Warner
    • 1
  • Valerie Mizrahi
    • 1
    Email author
  1. 1.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 SciencesUniversity of Cape TownCape TownSouth Africa

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