Abstract

Synthetic biology aims to develop methods of genetic regulation not seen in nature to manipulate biological systems, allowing them to achieve incredibly complex feats of biotechnology. One genetic element which has been receiving some attention by synthetic biologists is a class of enzymes known as serine recombinases. These enzymes have been shown to catalyze integration, excision, and inversion of DNA in a site-specific and directional manner. Due to the variety of activities of these enzymes and their irreversible mechanism of action, these enzymes could be used to construct gene regulation devices analogous in function to the digital memory of modern computers. This “genetic memory” could potentially offer improvements to the field of metabolic engineering and industrial biotechnology where traditional methods of genetic induction may not be sophisticated enough to provide total regulation of each step of complex metabolic pathways, and which may be considered expensive when carried out in large scales. This thesis aims to explore the use of DNA inversion catalyzed by the serine integrase from the BXB1 mycobacteriophage as a method of gene control in the brewer’s yeast Saccharomyces cerevisiae, which is a common strain used in industrial biotechnology. An assay was developed wherein a yeast-enhanced Green Fluorescent Protein (yeGFP) coding sequence could potentially be inverted with respect to a promoter, allowing for the expression of the reporter to indicate whether integrase-mediated inversion has occurred. Three conditions were tested to determine the parameters of the BXB1 integrase inversion in yeast: One where the yeGFP sequence began in a reverse compliment orientation with respect to a promoter element on a low-copy plasmid, and upon inversion would result in a translationally functional “forward” orientation which would be detectable by GFP fluorometry. Another plasmid based assay where the yeGFP sequence began in the translationally functional forward orientation and would be inverted to a non-functional reverse compliment orientation was also constructed. Finally, a third assay where the yeGFP reporter would be integrated into a yeast chromosome in the reverse compliment orientation and could potentially be inverted into the translationally functional orientation upon expression of the BXB1 Integrase was also undertaken. The results from these assays show that this reporter system could be made to “activate” GFP fluorescence on a plasmid, but could not be found to inactivate GFP fluorescence on a plasmid or activate the chromosomally integrated GFP reporter. These results, call into serious question the utility of the BXB1 serine integrase, and possibly serine integrases in general, as tools for synthetic biology in Saccharomyces cerevisiae.