Cloning Metabolic Enzymes from Mycobacterium tuberculosis

Sequences encoding GSase (Rv1212c; glgA), ADP-Glc PPase (Rv1213; glgC), UDP-Glc PPase (Rv0993; galU) and Tre-6P Sase (Rv3490; otsA) from Mtb H37Rv were amplified by PCR using genomic DNA as the template. Genomic DNA was kindly provided by Drs. Marisa Romano and Fabiana Bigi, from INTA Castelar (Argentina). Primers are listed in Supplemental Table I and were designed for each gene using available genomic information [38] (link), [39] (link) in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html). PCR reaction mixtures (50 μl) contained 100 ng of genomic DNA, 2 pg of each primer; 0.2 mM of each dNTP; 2.5 mM Mg²⁺, 5% (v/v) DMSO and 1 U Pfu DNA polymerase (Fermentas). Standard conditions of PCR were used for 30 cycles: denaturation at 94 °C for 1 min; annealing at 74 °C for glgC, 71 °C for glgA and 70 °C for galU and otsA, for 30 s, and extension at 72 °C for 3 min, with a final extension of 10 min at 72 °C. PCR reaction mixtures were resolved in 1% (w/v) agarose gels and PCR products were purified by means of Wizard SV gel & PCR Clean Up kits (Promega). The amplified genes [previously treated with Taq polymerase (Fermentas) and dATP] were cloned into the T-tailed plasmid pGEM-TEasy.