Project description:Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe177Ser) and, similarly, Tyr195His, and Lys218Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods. Experiment Overall Design: We measured transcription levels for two strains (wild type control and mutant spt15) under normal (0% ethanol, 20 g/L glucose) and stress (5% ethanol, 60 g/L glucose) in biological triplicate.
Project description:We measured transcriptional changes in four strains – P2, rpoD3, rpoA14, and rpoA27 - in an effort to understand mechanisms by which L-tyrosine production is positively influenced by the presence of mutant rpoA- and rpoD-encoded transcriptional components. Strains P2, rpoA14, rpoA27, and rpoD3 were grown in 50 ml MOPS minimal medium to an OD600 of approximately 0.4. Triplicates samples of RNA (on three separate days) were then extracted for hybridization onto Affymetrix microarrays.