Theoretical Modeling and Experimental Investigation of Host-Plasmid Interactions in Recombinant Escherichia coli

Citation

Peretti, Steven William (1987) Theoretical Modeling and Experimental Investigation of Host-Plasmid Interactions in Recombinant Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/M1F6-R062. https://resolver.caltech.edu/CaltechETD:etd-03102008-130217

Abstract

Microbial metabolism has been mathematically represented with sufficient mechanistic rigor to allow consideration of the ramifications of introducing recombinant vectors on host cell metabolic activity. The model was first verified using details of transcription and translation sufficiently stringent to test the simulation of RNA polymerase equilibrium distribution on promoter regions and the activation of ribosomes by binding of mRNA.

Plasmids were added to the formulation, and the dependence of productivity and growth rate was simulated. The results show good agreement with those obtained by other researchers. This model structure was then used to simulate potentially beneficial metabolic engineering scenarios in an attempt to identify those processes limiting productivity for recombinant systems. Simulation results indicate that transcription is the metabolic bottleneck that limits product synthesis. In addition, the most efficient strategy for enhancing plasmid product synthesis was shown to be increasing the efficiency of plasmid mRNA translation.

An asynchronous population was simulated in order to study the transient behavior of the cell model. Based on plasmid-free simulation results, the model accurately reflects the predominant metabolic structure for control of macromolecular synthesis. Transient response considerations for plasmid-containing populations suggest that dynamic reactor operation, in the form of induction of plasmid promoter activity, leads to transient accumulation of product which exceeds that attained during the subsequent balanced growth.

Finally, the effect of different plasmid copy numbers and of the concommitant expression of a constitutive plasmid-borne gene were investigated experimentally. Radio-labeling techniques combined with filter hybridizations were used to study the transcription rate from the β-lactamase promoter as well as the level of corresponding mRNA present in the cell. Coupled with product activity measurements, the stability of the plasmid-derived message, as well as the efficiency of its translation into protein are reduced significantly as copy number increases.

Thesis Files