SGER: Advanced Ty-Mediated Integration Methods for Metabolic Engineering and Protein Production in Yeast

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9703527 DaSilva The use of Saccharomyces cerevisiaae to produce proteins is limited by the narrow choice of vectors available for gene delivery. Currently, plasmids can be employed to introduce single genes at low copy number. When multiple copies are delivered by delta mediated delivery, the multiple sets are inserted in tandem leading to instability. There is no method available currently that can be used for the controlled addition of multiple genes or multiple, non- tandem copies to S. cerevisiae. This SGER proposal will test the feasibility of using yeast transposes, mobile segments of DNA that replicate by a retrovirus-like mechanism, to act as vectors for foreign genes. A cassette will be constructed that contains the gene of interest (in this case the gene for beta- galactosidase for ease of assay) plus a selection gene that can be used to select transposed yeast cells and latter deleted by a "popping out" mechanism so the cassette and same marker can be used to insert another gene. The URA3 gene flanked by two direct bacterial his G repeats will be used as the on-off switch. The transposed cells will be selected on uracil-deficient plates and then the cells that have been "popped-out" will be selected on 5-FOA which allows only those cells without the URA3 gene to grow. ***