Mtb escape mutant sequencing

Mtb strains were genetically engineered with up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline (dual-lysin strain), and a degron domain (dd)-NadE fusion which induces ClpC1-dependent degradation of the essential enzyme NadE negatively regulated by trimethoprim (ddTMP strain). Escape strains of dual-lysin kill switch were raised in the abscence of tetracycline, and escape strains of ddTMP kill switch were raised in the absence of trimethoprim. Similarly, escape mutants of the combined triple kill switch strain were generated after depletion of both trimethoprim and tetracycline. The escape mutants are sequenced to understand genetic determinants of resistance.