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.本研究中的结核分枝杆菌（Mtb）菌株经基因工程改造，搭载最多3种致死开关：一是受四环素负调控的双份分枝杆菌噬菌体溶素操纵子（双溶素菌株）；二是降解域（degron domain，dd）-NadE融合编码序列，该序列可介导依赖ClpC1的必需酶NadE降解，且该开关受甲氧苄啶负调控（ddTMP菌株）。 双溶素致死开关的逃逸菌株是在无四环素的培养条件下筛选获得，ddTMP致死开关的逃逸菌株则在无甲氧苄啶的培养条件下得到。同理，搭载组合型三重致死开关的菌株，其逃逸突变体可在同时耗尽甲氧苄啶与四环素后诱导生成。目前已对这些逃逸突变体完成测序，以解析介导耐药性的遗传决定因子。