The generation of conditional mutants has been an effective approach to studying bacteria and validating drug targets, and mutants of Mycobacteria are no exception. However unlike other bacteria, there is still a paucity of available tools for Mycobacteria. We constructed a new plasmid containing tetracycline-repressive expression system (TetRr1.7) and Xer Site-Specific recombinase system to generate label-free controllable expression strains. The plasmid was subsequently used to construct a strain of M. tuberculosis expressing the only copy of d-alanine:d-alanine ligase under the control of the tetracycline-repressive promoter. The results showed that the mutant strain lost the ability of colony formation, became more sensitive to d-cycloserine and the cell wall of the mutant strain was disrupted when anhydrotetracycline was added to the medium. Taken together these observations, confirmed that the expression of d-alanine:d-alanine ligase was tightly controlled by the promoter. In conclusion, the new plasmid is a convenient tool for constructing stable conditional mutant strains in Mycobacteria and can be used for future target identification.