Identification of the translational start site of mCherry in M. tuberculosis . Plasmids carrying mCherry were electroporated into M. tuberculosis and transformants selected on solid medium using hygromycin. The predicted proteins expressed from each plasmid are - pCherry29 = mCherry226; pCherry30 = mCherry219; pCherry0 (control plasmid) = no mCherry expression. (A) Transformant colonies. (B) Fluorescence was measured in liquid culture. Cultures were measured at Ex587/Em610 and results are expressed as relative fluorescence units (fluorescence/OD). Data are the mean and standard deviation from three independent transformants (C) Western analysis of protein expression in E. coli. Cell-free extracts were generated from transformants carrying plasmids and probed with anti-mCherry antibodies. Lane 1: no plasmid. Lane 2- pCherry10 (mCherry235). Lane 3- pCherry29 (mCherry226). Lane 4- pCherry30 (mCherry219). A non-specific band reacting with the commercial antibody was seen in all lanes, including E. coli lacking a plasmid.