Generating Bait Strains for Yeast One-Hybrid Assays
Program in Systems Biology; Program in Molecular Medicine; UMass Metabolic Network
Genetics and Genomics | Laboratory and Basic Science Research | Molecular Biology | Systems Biology
Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) "preys" can bind a DNA fragment of interest that is used as the "bait." Undertaking Y1H assays requires the generation of a yeast "bait strain" for each DNA fragment of interest that features the DNA bait coupled to a reporter(s). Plasmids encoding TFs fused to the Gal4 activation domain (AD) are then introduced into the bait strain, and activation of the reporter(s) indicates that a TF-DNA interaction has occurred. Here, we present a protocol for the first part of the strategy-the generation of a bait strain for Y1H assays. We assume that the DNA bait has already been cloned into two different reporter constructs: One places the fragment of interest upstream of HIS3, an auxotrophic growth marker, whereas the other places the DNA bait upstream of LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, generation of the bait strain involves using homologous recombination to integrate the two reporters into the genome of the yeast strain, screening individual integrants for background reporter expression (i.e., expression in the absence of a TF), and using polymerase chain reaction (PCR) and sequencing to confirm the DNA bait identity from both integrated reporter cassettes.
DOI of Published Version
Cold Spring Harb Protoc. 2016 Dec 1;2016(12):pdb.prot088948. doi: 10.1101/pdb.prot088948 Link to article on publisher's site
Cold Spring Harbor protocols
Fuxman Bass, Juan; Reece-Hoyes, John S.; and Walhout, Albertha J. M., "Generating Bait Strains for Yeast One-Hybrid Assays" (2016). Program in Systems Biology Publications and Presentations. 107.