Y190 Chemically Competent Cell

Y190 Chemically Competent Cell

The Y190 strain is a yeast strain developed by Clontech for the GAL4 system used in yeast two-hybrid experiments. It is of the MATa type and can either be directly transformed with plasmids or mated with the MATα type yeast strain Y187 for protein interaction validation or library screening experiments. The transformation markers are: trp1, leu2, cyh2; the reporter genes are: lacZ, HIS3. The Y190-GAL4 yeast two-hybrid system requires the use of two compatible plasmids: (pGB and pACT2) or (pGBKT7 and pGADT7). The pGB plasmid is derived from pGBKT7, with a selection marker of TRP1, and is used to express a fusion protein of the DNA-BD (from amino acids 1 to 174 of the yeast transcription factor GAL4) and the target protein (Bait); the pACT2 plasmid is structurally and functionally similar to pGADT7, with a selection marker of LEU, and is used to express a fusion protein of the AD (GAL4 amino acids 768 to 881) and the target protein (Prey). The principle of the GAL4 system is that a complete yeast transcription factor GAL4 can be divided into two functionally independent domains: the DNA-binding domain (DNA-BD) located in the N-terminal 1 to 174 amino acid segment and the transcription activation domain (AD) located in the C-terminal 768 to 881 amino acid segment. The DNA-BD can recognize the upstream activation sequence (UAS) of GAL4-responsive genes and bind to it. The AD can initiate the transcription of genes downstream of the UAS. BD and AD cannot activate transcription when they are separate, but when they are brought close together, they exhibit full GAL4 activity, which activates transcription of the downstream genes of promoters containing UAS. Under normal conditions, BD does not bind to AD, and the proteins to be tested are separately fused to BD and AD, forming the bait fusion protein (bait-BD) and prey fusion protein (prey-AD). If there is interaction between bait and prey, it will promote the proximity of BD and AD, forming complete GAL4, thereby activating the transcription of the reporter gene. The Y190 competent cells are specially processed and can be stored at -80°C for 12 months, and the pGADT7 plasmid (7988 bp, Amp^R) has a transformation efficiency of >10⁴ cfu/μg DNA.

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