Y187 Chemically Competent Cell

Y187 Chemically Competent Cell

The Y187 strain is a single and double hybrid experimental yeast strain developed using the GAL4 system by Clontech, of MATα type, which can be directly transformed with plasmids or screened through mating operations with MATa type yeast strains (Y2HGold, AH109, etc.). The transformation markers are: trp1, leu2, and the report genes are: lacZ, MEL1. The Y187-GAL4 yeast double hybrid system requires the use of two plasmids: pGBKT7 and pGADT7. The selection marker for plasmid pGBKT7 is TRP1, used for expressing the DNA-BD (from the yeast transcription factor GAL4 amino acids 1~174) fused with the target protein (Bait); the selection marker for plasmid pGADT7 is LEU, used for expressing the AD (GAL4 amino acids 768~881) fused with the target protein (Prey). The principle of the GAL4 system: a complete yeast transcription factor GAL4 can be divided into two structurally independent functional domains: the DNA-binding domain (DNA-BD) at the N-terminus (amino acids 1~174) and the transcription activation domain (AD) at the C-terminus (amino acids 768~881). The DNA-BD can recognize and bind to the upstream activating sequence (UAS) of GAL4-responsive genes. The AD can initiate transcription of the gene downstream of UAS. BD and AD cannot activate transcription independently, but when they are brought close together, they present the complete GAL4 activity, enabling the transcription of genes downstream of promoters containing UAS. Under normal conditions, BD does not bind with AD, and the proteins to be tested are fused with BD and AD separately, forming bait fusion protein (bait-BD) and prey fusion protein (prey-AD). If the bait and prey interact, it brings BD and AD closer together, forming a complete GAL4, thus activating the transcription of the reporter genes. Y187 has two reporter genes: lacZ and MEL1, which are driven by two different promoters (G1, M1). These two promoters share the same 17 bp core region recognized by GAL4, while the rest are different, greatly reducing the likelihood of false positives in the yeast double hybrid assay. The Y187 competent cells are prepared through a special process, and can be stored at -80°C for 12 months, with the pGADT7 plasmid (7988 bp, Amp^R) showing a transformation efficiency of >10⁴ cfu/μg DNA. 

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