Experiment Overview

Repository ID: FR-FCM-Z3J2 Experiment name: Expression of pTDH3-YFP in RAP1 mutant strains of yeast MIFlowCyt score: 24.75%
Primary researcher: Fabien Duveau PI/manager: Patricia Wittkopp Uploaded by: Fabien Duveau
Experiment dates: 2016-04-07 - 2016-04-08 Dataset uploaded: Mar 2021 Last updated: Mar 2021
Keywords: None Manuscripts:
Organizations: University of Michigan, Ann Arbor, MI (USA)
Purpose: The goal of this experiment is to quantify the effects of random and targeted mutations in the sequence of the RAP1 gene on expression of the reporter gene pTDH3-YFP (YFP expressed under control of the TDH3 promoter in yeast). RAP1 encodes a transcription factor known to directly bind the TDH3 promoter. The experiment includes 4 replicate populations of 483 strains with random mutations in RAP1 sequence and 10 genotypes with a mutation targeted to residues of the RAP1 protein expected to make direct contact with DNA (i.e. with the TDH3 promoter). For each sample, the fluorescence of at least 1000 cells was measured.
Conclusion: Random mutations in RAP1 coding sequence are very unlikely to alter the activity of the TDH3 promoter. Targeted mutagenesis suggest that this is because nonsynonymous mutations in key residues of RAP1 are lethal.
Comments: None
Funding: Not disclosed
Quality control: The experiment includes 36 replicate populations of a strain that does not contain the pTDH3-YFP reporter gene and that is used to estimate the level of autofluorescence. 108 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.
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