A useful gene cassette for conditional knock-down of essential genes by targeted promoter replacement in Mycobacteria

Abstract

A direct method to study essential genes is to construct conditional knock-down mutants by replacement of their native promoter by an inducible one. In Mycobacteria, replacement of an essential gene promoter with an anhydrotetracycline inducible one was successfully used but required a multi-step approach. In this work, we describe a gene cassette for the engineering of a conditional knock-down mutant, which allows the one-step targeted replacement of mycobacterial promoters by an anhydrotetracycline-inducible promoter. The functionality of this cassette was successfully tested by engineering conditional clpP and SecA1 mutants of Mycobacterium smegmatis.

Keywords

• ClpP
• Mycobacterium smegmatis
• promoter replacement
• recombineering
• SecA1
• tetracycline promoter