M. tuberculosis strain H37Rv (ATCC27294) was inoculated in Lowenstein-Jensen slant from frozen stock and incubated at 37°C for 2 weeks, and then transferred and grown in 100 mL Middlebrook 7H9 broth supplemented with 0.05% Tween 80, 0.2% glycerol, and OADC (Oleic Albumin Dextrose Catalase) for another 2 weeks. The culture was cooled on ice for 5 min and collected by centrifugation. The pellets were washed twice with 30 mL 10% precooled glycerol and resuspended in 5 mL 10% cold glycerol. For electroporation, 1 μg recombinant plasmid was mixed with 100 μL competent cells in 0.2 cm cuvette, and the transformation was conducted with the Gene Pulser Xcell Electroporation Systems (Bio-Rad) with the following conditions: 2.5 kV, 1,000 Ω, and 25 μF. After shock, 1 mL of 7H9 broth supplemented with OADC was immediately added into the cuvette. The culture was incubated for 2 days at 37°C and plated on Middlebrook 7H10 agar supplemented with OADC, 20 μg/mL kanamycin and 100 ng/mL anhydrous tetracycline. The plates were sealed by parafilm and incubated for 20 to 30 days at 37°C. M. smegmatis strain mc 2 155 (ATCC700084) was inoculated in7H10 with 10% ADC enrichment from frozen stock and incubated at 37 °C for 4 days. A single colony of a M. smegmatis strain was inoculated into 2 mL Middlebrook 7H9 broth supplemented with 0.05% Tween 80, 0.2% glycerol and ADC (Albumin Dextrose Catalase) at 37°C for 24 h. The cells were 1:100 diluted into 100 mL Middlebrook 7H9 broth supplemented with 0.05% Tween 80, 0.2% glycerol and ADC for another 12 to 15 hours. When the optical density at 600 nm of the culture reached 0.8 to 1, the culture was cooled on ice for 20 min and collected by centrifugation in 50-mL conical tubes at 4,000 rpm for 10 min. The pellets were washed twice with 30 mL 10% precooled glycerol and resuspended in 10 mL 10% cold glycerol. For electroporation, 100 ng plasmid was mixed with 100 μL competent cells in 0.2 cm cuvette, and the transformation was conducted with the Gene Pulser Xcell Electroporation Systems (Bio-Rad) with the following conditions: 2.5 kV, 1,000 Ω, and 25 μF. After shock, 1 mL of 7H9 broth supplemented with ADC was immediately added into the cuvette. The culture was incubated for 3 hours at 37°C and 10% portions of the culture were plated on Middlebrook 7H10 agar supplemented with ADC, 20 μg/mL kanamycin and 20 ng/mL anhydrous tetracycline. The plates were sealed by parafilm and incubated for 7 to 10 days at 37°C.