2.2 Strain and transformation
The ATCC 13032 C. glutamicum strain was obtained from the Korean Agricultural Culture Collection (KACC). C. glutamicum competent cells were prepared as previously described by Ruan et al. (2015).[32] Briefly, the cells were cultured in LHB solid media (20 g L-1 LB broth, 18.5 g L-1 brain heart infusion (BHI), 18 g L-1 agar). A single colony was inoculated into 5 mL BHI media (0.2 g L-1K2HPO4, 0.3 g L-1NaH2PO4, 0.5 g L-1MgSO4·7H2O, 10 g L-1(NH4)2SO4, 37 g L-1 BHI, pH 7.2) and cultured at 30 °C for 12 h. The microbes were harvested and re-cultured in 20 mL NCM (1.0 g L-1 yeast extract, 5 g L-1 tryptone, 5 g L-1 glucose, 0.3g L-1 trisodium citrate,17.4 g L-1K2HPO4, 0.05 g L-1MgSO4·7H2O, 91.1 g L-1sorbitol, 11.6 g L-1 NaCl, pH 7.2) at 30 °C for 4 h. After harvesting, the microbes were rinsed three times with ice-cold 10% glycerol. The microbes were then centrifuged and resuspended with 2 mL ice-cold 10% glycerol after which 90 μL of cells were aliquoted into microcentrifuge tubes. The competent cells were stored at -80 °C.
Plasmid DNA (5–10 μL) was added to 90 μL of competent cell solution and transferred to a 2 mm electroporation cuvette (Cat. No. Z706086-50EA, Sigma-Aldrich, USA). Electroporation was performed with a MicroPulser system (BioRad) 1.8 kV for 5 ms. After electroporation, 900 μL of liquid BHIS media (18 g L-1 BHI, 91 g L-1sorbitol) was added and resuspended, after which the cells were immediately incubated for 6–15 min at 46 °C. The cells were then plated on LBHIS (5 g L-1 tryptone, 5 g L-1NaCl, 2.5 g L-1 yeast extract, 18.5 g L-1 BHI, 91 g L-1 sorbitol, 18 g L-1 agar, pH 7.2) containing 50 μg mL-1 kanamycin or 30 μg mL-1streptomycin, and incubated at 30 °C until colonies appeared.