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.