Abstract
The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described that can be used to confirm gene essentiality using gene switching, following the construction of a delinquent strain. Because deletion mutants cannot be created for essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal gene copy to be deleted. The integrated vector can then be replaced using the gene switching method; where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm functionality of gene homologues and to easily identify essential operon members.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Brown, A.C. (2009). Gene Switching and Essentiality Testing. In: Parish, T., Brown, A. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 465. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-207-6_23
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DOI: https://doi.org/10.1007/978-1-59745-207-6_23
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Publisher Name: Humana Press, Totowa, NJ
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