EBY100 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. EBY100 is a MATa strain for yeast surface display，which is used in combination with the pYD1 plasmid and can be directly transformed for protein display assays. Its transformation markers are leu2 and trp1. The EBY100-pYD1 yeast display system anchors target proteins on the cell surface via the Saccharomyces cerevisiae a-agglutinin receptor to facilitate subsequent screening. The basic principle is as follows: the a-agglutinin receptor consists of two subunits，AGA1 and AGA2. The AGA1 protein (725 amino acids) is synthesized by EBY100 cells，secreted extracellularly，and covalently bound to β-glucan in the yeast cell wall. The AGA2 protein (69 amino acids) can attach to AGA1 through two disulfide bonds. The target protein is constructed into the pYD1 plasmid for fusion expression with AGA2，with AGA2 at the N-terminus and the target protein at the C-terminus. When EBY100 harboring the pYD1 plasmid is induced in medium containing galactose and no glucose，it expresses the AGA2-target protein fusion protein. The fusion protein binds to cell-surface AGA1 via AGA2，thereby displaying the target protein on the yeast surface. The transformation efficiency of EBY100 competent cells detected with pGBKT7 plasmid (7303 bp，Kan?) is higher than 10? cfu/μg DNA. Genotype: MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-Δ200 his3-Δ200 pep4::HIS3 prbd1. 6R can1 GAL