Fig 8. Comparison between L5-tdTomato and Hsp60-tdTomato M. bovis BCG strains in protein levels, transcript numbers and gene copies of tdTomato.

A. Comparison of the fluorescence from supernatant of bacterial lysates between M. bovis BCG L5-tdTomato and Hsp60-tdTomato strains. M. bovis BCG L5-tdTomato and Hsp60-tdTomato strains were grown until OD₆₀₀ = 0.75. 10⁶ bacteria/ml were ultrasonically lysed in 1x PBS with 0.1% Triton X-100. After centrifugation, the supernatant from the samples was compared using a multimode reader with 530 nm excitation and 590 nm emission wavelengths. B. Western Blotting to compare tdTomato protein levels expressed from an L5 promoter with that expressed from the Hsp60 promoter. The strain carrying the Hsp60 vector was used as a negative control. Bacterial housekeeping gene GroEL2 was used as the loading control. C. Quantification of relative band intensities in D by densitometry. D. Real time RT-PCR to compare transcripts of tdTomato expressed from the L5 promoter with transcripts levels expressed from the Hsp60 promoter. E. Real time q-PCR to compare copy numbers of tdTomato expressing plasmid with the L5 promoter and that carrying the Hsp60 promoter. Data represent one of three replicate experiments. Student’s t-test was performed to examine difference between the L5-tdTomato and Hsp60-tdTomato strains in fluorescence of extracted protein, transcript numbers and gene copies. * represents P<0.05; and ** represents P<0.01.