Figure 1.
TAT-survivin (T34A) expression in E. coli BL21 (DE3) under acetate stress in shake flasks and bioreactors.
A) and B), TAT-survivin (T34A) expression and cell growth of E. coli BL21 (DE3) under different conditions of acetate stress in shake flasks. The experiments were conducted in 50 mL of YTA-glucose medium (pH 7.5). C) and D), The profiles of TAT-survivin (T34A) expression, cell growth and acetate production in a 30-L bioreactor. The strategy of maintaining glucose at 10±5 g/L led to a constant increase of medium acetate from 10 h. E) The DO profile of the 30-L bioreactor cultivation from 10 h when IPTG was added (acetate: 110–250 mM). F) The DO profile of the 4-L bioreactor cultivation from 4 h when IPTG was added (acetate: 5–30 mM). In both bioreactors E. coli BL21 (DE3) cells were cultivated in YTA medium, but induced at different levels of acetate.
Figure 2.
Effect of pH on cell growth of E. coli BL21 (DE3) in BYT-glycerol medium containing NaCl (A) and acetate (B).
Figure 3.
Intracellular acetate accumulation in E. coli BL21 (DE3) cells under acetate stress at 50, 100, 200 and 300 mM in BYT-glycerol medium at pH 6.5 (A) and 7.5 (B).
C) Ratio of intracellular acetate concentration at pH 6.5 vs. pH 7.5.
Figure 4.
Effect of alkaline pHs on the cell growth of E. coli BL21 (DE3).
A) Curves of cell growth over the 4-h cultivation at pH 6.5, 7.0, 7.5, 8.0 and 8.5. B) The specific cell growth rates at various pHs. The specific cell growth rates were determined from the cell growth curves over the first 3 h of cultivation in 20 mL of BYT-glycerol medium in 250-mL shake flasks.
Figure 5.
Effect of alkaline pHs on recombinant protein expression by E. coli BL21 (DE3).
A) GST, B) CYP and C) GFP. The protein expression was conducted at pH 6.5, 7.5 and 8.5 in 20 mL of BYT-glycerol medium in 250-mL shake flasks as described in Methods and Materials.
Figure 6.
Comparison of the pH effect on recombinant protein expression by E. coli BL21 (DE3) grown on 300 mM NaAc.
A) GST, B) CYP and C) GFP. The protein expression was conducted at pH 6.5 and 7.5 in 20 mL of BYT-glycerol medium in 250-mL shake flasks. C, BYT-glycerol medium containing 300 mM NaCl as control; Ac, BYT-glycerol medium containing 300 mM NaAc.
Figure 7.
Alleviation of acetate-caused cell growth inhibition by alkaline pH shift from pH 6.5 to pH 8.0 at various temperatures.
E. coli strains A) BL21 (DE3), B) Origami (DE3) and C) DH5α were tested. Specific growth rates were determined in 20 mL of BYT-glycerol medium containing 300 mM NaCl or NaAc in 250-mL shake flasks.
Figure 8.
Effects of pH on the MTT-reducing activity of the E. coli cell membrane under acetate stress.
E. coli BL21 (DE3) cells were investigated on 0, 50, 100 and 300 mM NaCl (A) or NaAc (B) in BYT-glycerol medium at pH 6.5, 7.0, 7.5 and 8.0.
Figure 9.
A schematic representation of the decrease in concentration of extracellular harmful acetic acid molecules (HAc) and intracellular acetate ions (Ac–) produced by an alkaline pH shift.
A) Diagram to show HAc formation from Ac– and diffusion across the cell membrane as an uncoupler. Ac– cannot permeate the E. coli cell membrane. B) Release of intracellular acetate by an alkaline extracellular pH shift from 6.5 to a higher pH. Dark blue indicates higher intracellular acetate concentrations. pKa, the dissociation constant of acetic acid.