Expression of pTDH3-YFP in GCR1 mutant strains of yeast

The goal of this experiment is to quantify the effects of random mutations in the coding sequence of the GCR1 gene on expression of the reporter gene pTDH3-YFP (YFP expressed under control of the TDH3 promoter in yeast). GCR1 encodes a transcription factor known to directly bind the TDH3 promoter. The experiment includes 4 replicate populations of 683 strains with random mutations in GCR1 sequence. For each sample, the fluorescence of at least 1000 cells was measured. Conclusion: Random mutations in GCR1 are likely to alter the activity of the TDH3 promoter (38% of mutants showed a decreased pTDH3-YFP expression by more than 3%). Null alleles of GCR1 decrease expression of pTDH3-YFP by 80% relative to a strain with wild type GCR1 sequence. The experiment includes 96 replicate populations of a strain that does not contain the pTDH3-YFP reporter gene and that is used to estimate the level of autofluorescence. 204 samples correspond to replicate populations of the wild-type progenitor strain used as a reference to quantify expression changes in each mutant. An additional 24 wild-type strains expressing pTDH3-YFP were arrayed at predetermined positions in each 96-well plates to correct for technical variation between plates or flow cytometry runs.