Plasmid Propagation in the Yeast Saccharomyces cerevisiae: Flow Cytometry Studies and Segregated Modeling

Citation

Wittrup, Karl Dane (1989) Plasmid Propagation in the Yeast Saccharomyces cerevisiae: Flow Cytometry Studies and Segregated Modeling. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/KQYB-CX46. https://resolver.caltech.edu/CaltechETD:etd-02212007-131607

Abstract

The baker's yeast Saccharomyces cerevisiae is potentially a very useful host for the production of pharmaceutical proteins by recombinant DNA technology. One requirement for efficient overproduction of a foreign protein in yeast is a stable recombinant DNA vector which is maintained at a high number of copies per cell. The rational design of such vectors requires knowledge concerning their propagation in a cell population. The purpose of this work is to develop mathematical and experimental tools for the study of multicopy plasmid propagation, and to apply these tools to the investigation of a particular type of yeast vector: a conditional centromere plasmid.

A method for measuring the distribution of plasmid copy number in yeast populations was developed, using β-galactosidase activity as a marker for plasmid copy number. Enzyme activity is assayed at the single-cell level using a fluorogenic substrate and flow cytometry. The relationship between single-cell fluorescence and enzyme activity is described by a mathematical reaction-diffusion model.

A segregated mathematical modeling framework was established to link measured copy number distributions with probabilistic models of single-cell plasmid replication and partitioning. Simplifications of the general integral-partial differential population balance equations were obtained for a discrete state variable, resulting in a linear system of ordinary differential equations.

Flow cytometry and segregated modeling were applied to the study of a conditional centromere plasmid. This type of plasmid can be amplified to high copy number by unequal partitioning, but the amplified copy number state is unstable in the absence of selection pressure. A segregated model of this plasmid's propagation was shown to be consistent with experimental observations. The conceptual model of plasmid instability suggests changes in the attributes of the host cell and plasmid construction to improve stability at high copy number. A segregated mathematical model of this type is necessary for the design of bioreactor operating conditions that optimize productivity.

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