I have cloned the wild type sequence of gyrB or rpsL gene of Mtb from strain H37Ra into PVV16 vector and transform the plasmid to mc2 155 (Segmentis strain).

However, for overexpression of gyrB, the MIC of ofloxacin is unchanged compared with Msm with PVV16 vector + no insert as control.

For overexpression of rpsL, the MIC of streptomycin is unchanged compared with Msm with PVV16 vector + no insert as control.

I have sequenced the plasmid and sure that I have already cloned the correct sequence into the vector.

Shall I do a realtime PCR to check the transcript level?

Probably we will not do any western blot because no antibody is available soon and the problem of cost