Flow cytometric analysis of recombinant Saccharomyces cerevisiae populations
Abstract
The objective of this research was the development of flow cytometric techniques to investigate the changes in the physiological characteristics of host Saccharomyces cerevisiae cells brought on by the introduction and subsequent expression of recombinant DNA. Two recombinant model systems were considered. The first system consisted of two recombinant strains containing the yeast SUC2 gene coding for the enzyme invertase, while the second model system consisted of two recombinant strains containing human HLZ gene coding for the enzyme lysozyme. Total protein content, cell size, and DNA content properties were chosen for the analysis of these systems because of their importance to the yeast cell cycle and cloned-gene expression. Cell cycle phase distributions were extracted from yeast DNA histograms through the development of a modified Dean and Jett model. The modification was required to overcome the broad coefficients of variation of these histograms caused by the heterogeneous nature of yeast DNA. The distributions of the cell within the cell cycle allowed for a further comparison of recombinant and host strains. It was observed that recombinant strains become larger in cell size and contained more total protein than host strains grown under similar conditions. There was also a decline in these levels during the second phase growth on ethanol. This decline was not detected in the host strain which suggested the inability of the recombinant host to handle the expression of the cloned-gene product at this stage of the fermentation. The degree of reduction in cell size and protein content was related to the amount of cloned-gene product produced. Cell cycle phase distribution analysis of the stationary and enthanol phases showed an increase in the number of cells located in the G$\sb2$ + M phase of the cell cycle. This was not observed for the host strain. Due to the secretory nature of the cloned-gene products considered in this research, it was hypothesized that this increase was due to a saturation of the yeast secretion pathway. This saturation was thought to be caused by the combination of the overexpression of the recombinant product and decrease in cell growth rate. (Abstract shortened with permission of author.)
Degree
Ph.D.
Advisors
Seo, Purdue University.
Subject Area
Chemical engineering|Biochemistry|Cellular biology
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