Fig. 4.

Cultivation parameters of four representative E. coli BL21(DE3) clones of Type A and Type B behavior expressing BSLA under inducing conditions. Characterization of four E. coli BL21(DE3) clones belonging to respiration behavior Type A [Arg107-CGT (WT), Leu143-TTG; white background] or respiration behavior Type B (Arg107-CGG, Leu143-CTG; grey background) during cultivation in Wilms-MOPS mineral autoinduction medium containing 0.5 g/L glucose, 5 g/L glycerol, 2 g/L lactose. Cultivation parameters: oxygen transfer rate (OTR, determined using a RAMOS device), biomass (scattered light), soluble product formation (fluorescence), dissolved oxygen tension (DOT, all determined using a BioLector device), carbon source concentrations, optical density (OD), pH-value, enzyme activity and protein content of the target protein per total protein of the cell (all determined from parallel experiments in conventional shake flasks). The red and orange dotted lines represent the expected depletion of glycerol and lactose, respectively, based on the OTR profile. The vertical grey dotted lines separate the five cultivation phases (I–V) identified by the OTR curves according to Rahmen et al. [23]. Cultivation conditions: 37 °C, 250 mL flasks, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm (in RAMOS and conventional flasks); 37 °C, 48-well Flowerplate, filling volume 1 mL, shaking frequency 1000 rpm, shaking diameter 3 mm (in BioLector)