E coli rosetta 2 de3 singles competent cells

The gene encoding P. aeruginosa MetRS was obtained through PCR amplification (MJ Mini Thermo Cycler, Bio-Rad, Hercules, CA) using P. aeruginosa PAO1 (ATCC 47085) genomic DNA as a substrate. A forward primer (5′-ATATGCTAGCTCCGAACCACGCAAGATC-3′), designed to add an NheI restriction site to the 5′ end of the gene and the reverse primer (5′-CTCTAAGCTTTTACTTGACGCGCTGGC-3′) which was designed to add a HindIII restriction site to the 3′ end of the gene, were used in the PCR. The PCR product was inserted into a pET-28b(+) plasmid (Novagen) digested with NheI/HindIII. The recombinant plasmid was transformed into E. coli Rosetta 2(DE3) Singles Competent Cells (EMD Millipore, Danvers, MA).
The E. coli bacterial cultures were grown in Terrific Broth containing 25 μg/mL of kanamycin and 50 μg/mL of chloramphenicol at a temperature of 37 °C to an optical density (A₆₀₀) of 0.6–0.8. The over-expression of P. aeruginosa MetRS in the cultures was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.25 mM. Growth of the culture was continued for 2 hours post-induction, and cells were harvested using centrifugation (10,000 g, 30 min, 4 °C). Fraction I lysates were prepared as previously described [7 (link)]. P. aeruginosa MetRS was purified to more than 98% homogeneity as previously described [6 (link)].

The gene encoding P. aeruginosa LeuRS was obtained through PCR amplification (MJ Mini Thermo Cycler, Bio­Rad, Hercules, CA) using P. aeruginosa PAO1 (ATCC 47085) genomic DNA as a substrate. A forward primer (5’-gaacgctagccacgagcaatatcagccact-3’), designed to add an Nhel restriction site to the 5’ end of the gene, and a reverse primer (5’-ggataagctttcagttggcgacgatgttg −3’), which was designed to add a HindIII restriction site to the 3’ end of the gene, were used in PCR. The PCR product was inserted into a pET-28b(+) plasmid (Novagen) digested with NheI/HindIII. This resulted in the gene, encoding P. aeruginosa LeuRS, fused to an upstream region encoding six histidine residues. The recombinant plasmid was transformed into E. coli Rosetta 2(DE3) Singles Competent Cells (EMD Millipore, Danvers, MA). Bacterial cultures were grown, expression of the target protein induced and P. aeruginosa LeuRS was purified as described [10 (link)].