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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