Y2HGold Chemically Competent Cell

Y2HGold Chemically Competent Cell

The Y2HGold strain is a yeast two-hybrid strain developed by Clontech for the GAL4 system, of MATa type, which can be directly transformed with plasmids or interact with MATα type yeast strain Y187 through mating operations for protein interaction validation or library screening tests. The transformation markers are: trp1, leu2, and the reporter genes are: AbAr, HIS3, ADE2, MEL1. The Y2HGold-GAL4 yeast two-hybrid system requires the use of two matching plasmids: pGBKT7 and pGADT7. The selection marker for the plasmid pGBKT7 is TRP1, used for expressing the fusion protein of DNA-BD (from the yeast transcription factor GAL4, amino acids 1 to 174) and the target protein (Bait); the selection marker for the plasmid pGADT7 is LEU, used for expressing the fusion protein of AD (GAL4, amino acids 768 to 881) and the target protein (Prey). The principle of the GAL4 system: a complete yeast transcription factor GAL4 can be functionally divided into two structurally independent domains: the DNA binding domain (DNA-BD) located at the N-terminal amino acid region 1 to 174, and the transcription activation domain (AD) located at the C-terminal amino acid region 768 to 881. The DNA-BD can recognize the upstream activation sequence (UAS) of GAL4-responsive genes and binds to it. The AD can initiate transcription of the genes downstream of UAS. BD and AD cannot activate transcription when they exist separately, but when they come close together, they exhibit full GAL4 activity, enabling transcription of downstream genes that contain UAS in their promoters. Under normal conditions, BD does not bind with AD, and the proteins to be detected are fused with BD and AD, forming bait fusion proteins (bait-BD) and prey fusion proteins (prey-AD). If bait and prey interact, it will promote the approach of BD and AD, forming complete GAL4, thus activating transcription of the reporter gene. Y2HGold has four reporter genes: AbAr, HIS3, ADE2, MEL1, each started by three different promoters (G1, G2, M1). These three promoters have the same 17 bp core region that GAL4 recognizes, while the rest is different, greatly reducing the probability of false positives in yeast two-hybrid assays. Additionally, the new reporter gene AbAr has the advantage of lower background compared to previous nutrient-deficient reporter genes, which also lowers the probability of false positives in yeast two-hybrid assays. Y2HGold competent cells are prepared using special processes and can be stored at -80°C for 12 months, with pGADT7 plasmids (7988 bp, Amp^R) exhibiting a transformation efficiency of > 10⁴ cfu/μg DNA. 

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