Expression and Culture of Mycobacterial Proteins

Escherichia coli Rosetta2 containing plasmid pET30b-Hsp18 (strain TPS681) or pDest17-MUL_3720 (strain TPS682) was grown at 37 °C in Luria-Bertani (LB) broth (Difco, Becton Dickinson, MD, USA) supplemented with 100 µg/ml ampicillin (Sigma-Aldrich, USA) or 50 µg/ml kanamycin to express 6xHIS-tagged Hsp18 or MUL_3720 recombinant protein. Mycobacterium ulcerans (strain Mu_1G897 from French Guiana (De Gentile et al., 1992 (link))) was grown at 30 °C in 7H9 broth or 7H10 agar (Middlebrook, Becton Dickinson, MD, USA) supplemented with oleic acid, albumin, dextrose and catalase growth supplement (OADC) (Middlebrook, Becton Dickinson, MD, USA), and 0.5% glycerol (v/v). M. bovis BCG (strain Sanofi Pasteur) used for vaccinations was grown at 37 °C in 7H9 broth or 7H10 agar supplemented with OADC. Mycobacterial colony counts from cultures or tissue specimens were performed using spot plating as previously described (Wallace et al., 2017 (link)).