I have Cloned a Mycobacteraial gene in pST-KT Anhydrotetracycline(ATc) inducible vector and electroporated in M. smagmatis. Transformants grown till OD 0.6-0.7 and 200ng/ml ATc were added and induced for 36 Hr at 37 C 200 RPM. The culture further cetrifuged at 12000 rpm for 10 min. Pellet resuspended in Resuspension Buffer and cells were ruptured by beat beating for 30 sec.×2 after adding glass beads. Sample centrifuged at 12000rpm for 10 min and soup & pellet loaded on 10% SDS gel. There was no induction seen afer CB staining. Also i have proceeded for Western blotting analysis using His tag Antibody, but nothing was seened in WB. Please guide me in this regards.

I have also tried to induce by tetracycline 100ng/ml but wasn't there.

Thank you