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Mycobacteria Protocols pp 131–149Cite as

Targeted Gene Knockout and Essentiality Testing by Homologous Recombination

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Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  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 Sciences, University of Cape Town, Cape Town, South Africa

    Krishnamoorthy Gopinath, Digby F. Warner & Valerie Mizrahi

Authors
  1. Krishnamoorthy Gopinath
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  2. Digby F. Warner
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  3. Valerie Mizrahi
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Corresponding author

Correspondence to Valerie Mizrahi .

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Editors and Affiliations

  1. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    Tanya Parish

  2. Infectious Disease Research Inst (IDRI), Seattle, Washington, USA

    David M. Roberts

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Gopinath, K., Warner, D.F., Mizrahi, V. (2015). Targeted Gene Knockout and Essentiality Testing by Homologous Recombination. 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-2450-9_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2449-3

  • Online ISBN: 978-1-4939-2450-9

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