Figure 5

Fed-batch cultivation of AcrA-O1 producing E. coli with semi-defined glycerol medium using three different feed and induction strategies. Strategy A - filled symbols: Linear feed of glycerol and tryptone from time -3 to 0 h, pulse of IPTG (1 mM) and L-arabinose (2 g L^-1) at time 0 h, linear feed of glycerol, tryptone, L-rabinose (2.75 g L^-1 h^-1) and IPTG (80 μM h^-1) from time 0 to 15 h. Strategy B - open symbols: Pulse of glycerol, tryptone, L-arabinose (4 g L^-1) and IPTG (1 mM) at time 0 h, pulse of glycerol, tryptone and L-arabinose (4 g L^-1) at time 4 h. Strategy C - shaded symbols: Pulse of glycerol, yeast extract and tryptone at time -2.6 h; pulse of glycerol, tryptone, L-arabinose (10 g L^-1) and IPTG (1 mM) at time 0 h; linear feed of tryptone, L-arabinose (1.6 g L^-1 h^-1) and IPTG (10 μM h^-1) from time 0 to 15 h. (A) Logarithmic growth curve (circles) and time course of biomass concentrations (squares), time 0 h (broken line): induction with L-arabinose and IPTG. (B) Time course of AcrA and AcrA-O1 formation. Normalized total cell protein samples were analysed by Western blot with anti-AcrA antibodies, numbers indicate time after induction, SF: samples from LB shake flask cultures. Lane B: same sample of strategy B as in middle blot, analysed on a blot with samples of strategy C (shorter development time, non-relevant lanes removed). E. coli CLM24 (pMIK44, pGVXN64, pGVXN114).