Use of an E. coli pgi Knockout Strain as a Plasmid Producer

Cells were incubated overnight at 37 °C and 250 rpm and used to inoculate 250-mL baffled shake flasks containing 50 mL of complex medium (20 g/L glucose, 10 g/L bacto peptone, 10 g/L yeast extract, 3 g/L ammonium sulfate ((NH4)2SO4), 3.5 g/L potassium hydrogen phosphate (K2HPO4), 3.5 g/L potassium dihydrogen phosphate (KH 2PO4), 200 mg/L thiamine, 2 g/L magnesium sulfate (MgSO4), and 1 mL/L of a trace element solution [9]) with 30 μg/mL kanamycin, pH 7.0, at an initial optical density at 600 nm (OD600) of approximately 0.1. Plasmid quantitation Analytical chromatography was performed in an ÄKTApurifier10 system (GE H ealthcare), using a commercial Tricorn high-performance column with a 1.7 mL bed volume (SOURCE 15PHE 4.6/100 PE, GE Healthcare) and following a modification of the HIC-HPLC (high-performance liquid chromatography) method described by Diogo et al.