Figure 7.

Detailed characterization of four E. coli BL21(DE3) clones belonging to respiration behavior Type A and Type B cultivated under inducing conditions. Characterization of four E. coli BL21(DE3) clones belonging to respiration behavior Type A (expressing wild type BSLA and variants S56P and S167P) and respiration behavior Type B (BSLA variant K170E) during the cultivation in Wilms-MOPS mineral autoinduction medium. (A) Characteristic growth parameters: oxygen transfer rate (OTR), biomass and product formation, carbon source concentrations, copy number of plasmid per genome, pH-value, and protein content of the target protein per total protein of the cell. Type A: white background; Type B: grey background. The five cultivation phases (I-V) are identified by the OTR curves and are separated by the vertical grey dotted lines. (I) increase in OTR due to growth on glucose, the depletion of glucose corresponds with a small OTR drop (first grey dotted line); (II) increase in OTR to first peak (second grey dotted line) due to growth on glycerol; (III) slightly decreasing or constant OTR, respectively, due to product formation on lactose with parallel consumption of glycerol, the depletion of lactose roughly corresponds with the minimum of the OTR (third grey dotted line); (IV) increase in OTR to second peak (forth grey dotted line) due to growth on residual glycerol; (V) end of cultivation, the depletion of glycerol roughly corresponds with the sharp drop in OTR. (B) SDS-PAGE analysis showing soluble protein per sample volume, and (C) SDS-PAGE analysis showing insoluble protein per sample volume after 0, 3, 4.5, 6, 7.5, 9, 10.5, 13, 14.5, and 18 h of cultivation (target protein band is framed, M = protein marker). Cultivation conditions: 37°C, 250 mL flasks, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm (in RAMOS); 37°C, 48-well Flowerplate, filling volume 1 mL, shaking frequency 1500 rpm, shaking diameter 3 mm (in BioLector).