Conditional expression-specialized transduction essentiality test (CESTET) is a genetic tool used to determine essentiality of individual genes in Mycobacterium smegmatis. CESTET combines specialized transduction, a highly efficient gene knockout method, with the utility of the inducible acetamidase promoter. In a merodiploid strain containing a second integrated copy of an essential gene under the control of the acetamidase promoter, transductants (gene knockouts of the native chromosomal copy of a gene) are obtained only in the presence of acetamide in the selection medium. Furthermore, effects of loss of essential gene function can then be studied by growing the transductants in medium depleted of acetamide.