Y2HGold Chemically Competent Cell

For the first use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 min，then place it on ice immediately and store it at -20℃ after use for standby. Thaw Carrier DNA on ice before subsequent use，and re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles. Competent cells are best thawed on ice. The use of high-concentration plasmids for transformation can correspondingly reduce the amount of bacterial solution used for final plating. Saccharomyces cerevisiae is temperature-sensitive with an optimal growth temperature of 28℃-30℃，and temperatures above 31℃ will affect yeast growth. Yeast grows slower in dropout medium than in YPDA medium，and the more deficient components in the medium，the slower the yeast grows. Y2HGold is a MATa strain for GAL4-system yeast two-hybrid experiments. It can be directly transformed with plasmids，or mated with the MATα yeast strain Y187 for protein interaction verification and library screening assays. The transformation markers are trp1 and leu2，and the reporter genes include AbAr，HIS3，ADE2 and MEL1. The Y2HGold-GAL4 yeast two-hybrid system requires two matched plasmids: pGBKT7 and pGADT7. The pGBKT7 plasmid carries the TRP1 selection marker and is used to express the fusion protein of DNA-BD (derived from amino acids 1~174 at the N-terminus of the yeast transcription factor GAL4) and the bait protein; the pGADT7 plasmid has the LEU selection marker and is used to express the fusion protein of AD (GAL4 C-terminal amino acids 768~881) and the prey protein. Principle of the GAL4 system: The complete yeast transcription factor GAL4 consists of two functionally independent domains: the DNA-binding domain (DNA-BD) located at N-terminal amino acids 1~174，and the transcriptional activation domain (AD) located at C-terminal amino acids 768~881. DNA-BD can recognize and bind to the upstream activation sequence (UAS) of GAL4-responsive genes，while AD initiates the transcription of downstream genes under UAS. BD and AD cannot activate transcription when existing alone; only when they are brought into proximity can they restore full GAL4 activity and trigger the transcription of genes downstream of the UAS-containing promoter. Under normal conditions，BD does not bind to AD. The proteins to be tested are respectively fused with BD and AD to form bait fusion protein (bait-BD) and prey fusion protein (prey-AD). If the bait and prey interact，they bring BD and AD close to each other to reconstruct functional GAL4，thereby activating the transcription of reporter genes. Y2HGold contains four reporter genes AbAr，HIS3，ADE2 and MEL1，driven by three distinct promoters (G1，G2，M1). These promoters share only the 17 bp core region recognized by GAL4 and differ in other sequences，which greatly reduces the false-positive rate of yeast two-hybrid assays. In addition，the novel reporter gene AbAr exhibits lower background compared with traditional auxotrophic reporter genes，further lowering the probability of false positives. The transformation efficiency of Y2HGold competent cells detected with pGADT7 plasmid (7988 bp，Amp?) is higher than 10? cfu/μg DNA. Genotype: MATa，trp1-901，leu2-3，112，ura3-52，his3-200，gal4Δ，gal80Δ，LYS2::GAL1UAS-Gal1TATA-His3，GAL2UAS-Gal2TATA-Ade2，URA3::MEL1UAS-Mel1TATA AUR1-C MEL1