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28 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGY3.4. Determination of ClpC1 as the target protein of ecumicin by mutation The mutant resistant to ecumucin was selected (Fig. 4). Based on the relatively high level of selective resistance to ecumicin, four strains (M. tuberculosis ITR6, ITR7, ITR12, and ITR13) were selected for whole-genome sequencing. Compared with M. tuberculosis H37Rv, the selected mutants all harbored nonsynonymous mutations in clpC1 (Rv3596c), ppsC (Rv2933), and espG3 (Rv0289). All resistant strains had the same mutations to espG3 (GGT%u00a1AGT; G191S) and ppsC (CAG%u00a1TAG; Q695stop), but harbored one of three mutations in the N-terminal repeat II of ClpC1 that resulted in proline for leucine 96 or serine or phenylalanine for leucine 92. Figure 4. Work flow chart for generation of ecumicin-resistant M. tuberculosis, and identification of target protein3.5. Drug Affinity Responsive Target Stability (DARTS) assayTo further examine which of these potential targets actually binds ecumicin, recombinant E. coli BL21 isolates overexpressing M. tuberculosis wild-type and mutant ClpC1 and EspG3 were tested for drug affinity responsive target stability (DARTS). A lysate of each recombinant E. coli was exposed to ecumicin at