NMY51 Chemically Competent Cell

For the first use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 min and then place it on ice immediately. After use，store it at -20℃ for later use. Thaw Carrier DNA on ice before next use. Re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles. Competent cells are preferably thawed on ice. Transformation with high-concentration plasmids can correspondingly reduce the amount of bacterial liquid used for final plating. Saccharomyces cerevisiae is temperature-sensitive with a suitable growth temperature of 28℃-30℃，and temperatures above 31℃ will affect yeast growth. Yeast grows slower in defective medium than in YPDA medium; the more defective components the medium contains，the slower the yeast grows. NMY51 is a MATa haploid strain of Saccharomyces cerevisiae，specifically developed for the DUAL membrane split-ubiquitin yeast two-hybrid system，which is mainly applied to screen and verify interactions between native membrane proteins with an extremely low false-positive rate. The transformation markers of this strain are trp1 and leu2-3，and the reporter genes include HIS3，ADE2 and lacZ. Primary selective growth screening is performed via the auxotrophic reporter genes HIS3 and ADE2，followed by quantitative or semi-quantitative screening through β-galactosidase colorimetric assay of the lacZ reporter gene. Three independent reporter genes are regulated by different promoters，effectively reducing the probability of false positives. Ubiquitin is a small molecule consisting of 76 amino acid residues; as a degradation signaling molecule，ubiquitin can attach to the N-terminus of another protein and be recognized by ubiquitin-specific proteases (UBPs)，leading to the cleavage and dissociation of the linked protein. Ubiquitin can be artificially divided into two parts: the N-terminal fragment (Nub) and the C-terminal fragment (Cub). Firstly，the isoleucine at position 3 of wild-type Nub is mutated to glycine，converting NubI to NubG，which greatly reduces its affinity for Cub and prevents spontaneous self-association or close interaction between Cub and Nub. Secondly，the Cub fragment is fused with the synthetic LexA-VP16 transcriptional activator to form the fusion protein Cub-LexA-VP16. Under normal conditions，NubG does not bind to Cub，UBPs cannot recognize the separated ubiquitin，and the transcriptional activator will not be cleaved off. The target proteins to be tested are respectively fused with NubG and Cub to generate the bait fusion protein (bait-cub-LexA-VP16) and prey fusion protein (prey-NubG). If interaction occurs between bait and prey，it brings NubG close to Cub，which is then recognized by UBPs，resulting in the cleavage and release of LexA-VP16. The released transcription factor enters the nucleus and activates the transcription of downstream reporter genes. Four types of bait plasmids are applicable to this system: pBT3-N，pBT3-SUC，pBT3-STE and pBT3-C，all carrying the LEU selection marker; three prey plasmids are available: pPR3-C，pPR3-SUC and pPR3-STE，with Trp as the selection marker. The transformation efficiency of NMY51 competent cells detected with pGADT7 plasmid (7988bp，Amp?) is higher than 10? cfu/μg DNA. Genotype: MATa，his3Δ200，trp1-901，leu2-3，112，ade2，LYS2::(lexAop)4-HIS3，ura3::(lexAop)8-lacZ，ade2::(lexAop)8-ADE2，GAL4