Plasmid Instability and Plasmid Gene Expression in E. coli.

The laclq gene was cloned into an expression vector in order to provide more stringent control over gene expression. A new host strain required for experimental purposes was developed by P1 transduction. A method for the measurement of substrate affinity (Ks) for glucose was investigated. The method used a set of computer programs applied to batch culture data. The method did not show conclusively a difference in Ks between P+ and P- cells, or for cells induced for different levels of plasmid gene product expression. Possible improvements in the method are discussed. A consistent reduction in maximum specific growth rate (μmax) values for cells with increasing product expression was demonstrated. An unstructured unsegregated mathematical model was developed using Monod kinetics. The model was capable of accurate simulation of plasmid instability in chemostat culture. Such a model has not been used previously for accurate simulation of plasmid instability. The Ks values of P+ and P- cells were evaluated using mathematical modelling of chemostat experiments. Absolute values for substrate affinity were not determined but modelling demonstrated the existence of a substrate affinity difference and of its importance in attributing to culture instability. The importance of μmax of the two cell types and of copy number of P+ cells on culture instability were also demonstrated. Product expression levels from plasmid genes were measured in cells grown in chemostat culture. Gene product levels were shown to be positively correlated to the plasmid copy number values predicted by the mathematical model. Study of the mRNA ribosome binding sites and particularly the translation initiation codons revealed the possible reason for expression of tryptophanase from the tac promoter in uninduced cultures. The effect of this expression on the plasmids instability and possible methods for improving this instability are discussed