Mycobacterial Strain Engineering and Protein Expression

The E. coli strain BL21(DE3) (F- ompT hsdS_β(r_β-m_β) dcm gal (DE3) tonA) (Novagen) was used for all protein expression. E. coli K-12 Mach1 T1 cells (ΔrecA1398 endA1 tonA Φ80ΔlacM15 ΔlacX74 hsdR(r_k⁺m_k⁺); Invitrogen) were used for all cloning experiments. M. smegmatis mc²155 and M. tuberculosis H37Rv strains were both used for growth profiles. Experiments involving the virulent H37Rv strains of M. tuberculosis were conducted in a BSL-3 laboratory following institutionally approved protocols. M. tuberculosis H37Rv was also used for the plating efficiency assay to determine recovery of viable bacteria upon VapC-mt11 expression. M. smegmatis mc²155 was used for metabolic labeling. The vapC-mt11 (Rv1561; “mt” refers to M. tuberculosis) gene was cloned using M. tuberculosis H37Rv genomic DNA. The DNA sequences of PCR fragments used for cloning were confirmed by automated DNA sequence analysis. The VapC-mt11 coding region was cloned into the pET28a expression vector (EMD Millipore) and the arabinose inducible pBAD33 plasmid (ATCC) after the addition of 5′ NdeI and 3′ BamHI restriction enzyme sites by PCR. For expression in Mycobacteria, the VapC-mt11 coding region with PCR generated 5′ ClaI and 3′SalI restriction sites was cloned into the corresponding sites in the anhydrotetracycline (ATc) inducible vector pMC1s^{15 (link)}.