Characterization and modeling of the pH-regulated expression vector, pSM-10
Tolentino, Gerard Joaquin
Doctor of Philosophy
A new type of plasmid vector has been developed for Escherichia coli. Foreign gene expression can be altered by external pH changes. pSM-10 is the first generation of these plasmid constructs that utilizes the cadA operon for gene regulation. The growth conditions necessary to maximize this response were characterized. GJT-001, a $cadR\sp-$ strain of E. coli, was isolated that allowed study of pH regulation without requiring high lysine concentrations. Up to a 200-fold increase in specific activity of $\beta$-galactosidase was observed upon lowering the pH of the growth media from pH 8.0 to 5.5. Anaerobic growth conditions can independently induce high gene expression. Maximum activities are observed under a combination of low pH and anaerobic conditions. The pH-induction effect is fully reversible and provides a high degree of expression control. The highest levels of induction were observed in batch culture when pH- and anaerobic-step changes were introduced at low optical densities. Rich media is also necessary for highest expression. Further investigation revealed that a possible induction factor may be present in yeast extract. In addition, pH induction is limited at high optical densities due to an apparent inhibition by accumulated waste products, specifically acetate. A mathematical model was developed for $\beta$-galactosidase expression, incorporating the effects of growth rate, yeast extract consumption and acetate inhibition. From this model maximal batch productivities are best achieved under aerobic growth conditions that maximize growth rate and optical density and also minimizes the inhibition effect of acetate.
Chemical engineering; Biochemistry; Cell biology