| dc.description.abstract | Following the publication of the Saccharomyces cerevisiae (budding yeast) genome sequence, the yeast community compiled a near complete collection of deletion strains for every open reading frame (ORF). This library of deletion strains has been extensively used in genome-wide experiments in an effort to comprehensively annotate the whole yeast genome. A number of other yeast libraries with peptide tags or other useful modifications introduced to each ORF have since been developed and have proven to be a useful tool in the field of functional genomics and proteomics. Traditional cloning methods require individual genetic constructs and cloning events for each mutant strain. Using these methods, creating genome-wide libraries is extremely labour-intensive, expensive and subsequently only feasible for larger research groups with lots of resources. Herein, I developed a rapid tag switching system combining CRISPR Cas9 genetic engineering with high throughput yeast automation to generate custom libraries of yeast strains, with each strain encoding a different tagged protein, in as little as two weeks. These tags include fluorophores and a collection of conditional degrons strains. This flexible genetic modification method can be applied to any starting yeast library for the parallel insertion of multiple genetic elements and has potential for applications in cell biology and biotechnology. Using these yeast libraries, I take a systematic approach to investigating protein interactions and mis-localisation at the yeast centrosome, commonly known as the Spindle Pole Body (SPB). The SPB serves as a Microtubule Organizing Centre; it is embedded in the nuclear membrane where it nucleates spindle microtubules and serves as a signalling hub for cell cycle regulators. Notably, the SPB produces and regulates the mitotic spindle, a vital microtubule structure required for separating sister chromatids into daughter cells during mitosis. Using the Synthetic Physical Interactions assay, I systematically screened forced association of the yeast proteome pairwise to the SPB. My findings indicate the “once per cycle” duplication of the SPB can be subverted by specific forced interaction events. | en_US |
