Tetracycline inactivation in E. coli

Antibiotic susceptibility testing was performed in E. coli MegaX cells (Invitrogen) bearing the pZE21 expression vector with the tetracycline inactivating gene of interest. Minimum inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) procedures⁵⁹ using Mueller-Hinton broth with 50 μg/mL kanamycin and a range of chlortetracycline concentrations profiled via absorbance measurements at 600 nm (OD₆₀₀) at 45 minute intervals using the Synergy H1 microplate reader (Biotek Instruments, Inc) for 48 hours at 37°C.