Background The cornea is the transparent, outermost part of the eye which protects the fragile intraocular contents from mechanical and pathogenic perturbations. It consists of three distinct layers: a tear-bathed, stratified layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium.
A library of Hsp90 mutants will be constructed with ePCR. SDM mutants may be made if there are any particularly compelling regions that we can specifically identify to mutate. The rationale is that more promiscuous Hsp90, or more actively expressed Hsp90 (or non-degraded), will be able to buffer a larger amount of phenotypic variation than standard strains. This underlying abnormally large phenotypic diversity will be exposed in subsequent screens of growth under industrially relevant concentrations of sugar and ethanol. The rate of succesful mutants will be compared to standard random mutagenesis techniques. Particularly succesful mutants will be sequenced and undergo DNA microarray analysis, to determine the gene clusters up or downregulated in the mutants.
Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.
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Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/>.