The creation of a genome-wide yeast deletion collection revealed that there are over 1,000 protein-coding genes that are essential for yeast viability. The function of many of these essential genes has yet to be defined. In part this is due to the difficulty of utilizing essential genes in functional assays. The Tet-promoters Hughes collection (yTHC) from the Hughes Laboratory, University of Toronto, helps to overcome this barrier.
The yTHC collection contains 800 essential yeast genes for which expression is regulated by doxycycline. The endogenous promoter has been replaced with a Tet-titratable promoter in the genome. Thus, the expression of the gene can be switched off by the addition of doxycycline to the yeast’s growth medium.
Development of the Strains2
The yTHC mutant strains are provided in the haploid MATa strain R1158. This strain was created from the background strain BY4741 by a one-step integration of the tTA transactivator, under the control of the CMV promoter, at the URA3 locus. A plasmid carrying a kanR-tetO7-TATA cassette was then integrated into the genome replacing the endogenous promoter. The correct integration was verified by PCR across the junctions and again using gene specific primers.
Additionally, each strain was qualified by its growth phenotype on YPD with 10µg/ml doxycycline. Without doxycycline in the media the Tet-promoter is fully activated. Addition of doxycycline in a titratable manner allows for down regulation of the promoter until the gene of interest is no longer expressed at detectable levels. The optimal condition for each strain needs to be determined empirically.
Background Strain = R1158 (derived from By4741)
Genotype of wildtype R1158 - URA3::CMV-tTA MATa his3-1 leu2-0 met15-0
*Wildtype R1158 is provided in YPD, with addition of 15% glycerol following incubation
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Individual strains are provided as a live culture in a 2ml tube. Each tube contains YPD broth with G418 (200µg/ml) and is supplemented with 15% glycerol following incubation. We will ship your clone at room temperature via express delivery. Store the stock clone at 4°C for up to one week or -80°C indefinitely. The Yeast Tet-Promoter collection is provided in 96-well microtiter plates containing frozen glycerol stock cultures in YPD with G418 (200µg/ml) plus 15% glycerol. We ship collections frozen on dry ice and these can be maintained indefinitely at -80°C.
