GS115 Chemically Competent Cell

1.For first-time use of Carrier DNA，boil the tube containing Carrier DNA in boiling water for 5 min，then place it on ice immediately. After use，store it at -20 ℃ for later use. Thaw Carrier DNA on ice before next use. After 3-5 repeated freeze-thaw cycles，re-denature the Carrier DNA. 2.Transformation with high-concentration plasmids can correspondingly reduce the final bacterial amount used for plate spreading. 3. Increase the dosage of plasmids when transforming 2-3 types of plasmids simultaneously. 4.The optimal growth temperature for Pichia pastoris is 28℃ to 30℃; temperatures above 32℃ will inhibit yeast growth. GS115 is a Pichia pastoris strain used for recombinant protein expression. As a histidine auxotrophic strain，GS115 has a mutation at the genomic histidine dehydrogenase (His4) locus and cannot synthesize histidine independently; certain Pichia pastoris expression vectors carrying the HIS selection marker，such as pPIC3. 5K，pPIC9K and pAO815，can express the HIS4 gene to complement the genetic defect of GS115，allowing positive integrants to be screened on histidine-deficient medium，while other Pichia pastoris plasmids without the HIS selection marker，including pPICZ A，B，C and pPICZα A，B，C，can also be used for positive transformant screening with Zeocin or bleomycin hydrochloride. The phenotype of GS115 is His?，Mut?，where His? indicates that GS115 is a histidine auxotrophic strain unable to grow on histidine-deficient medium，and Mut? means GS115 carries a functional AOX1 gene; Pichia pastoris is a methylotrophic yeast capable of utilizing methanol as the sole carbon source，and the first step of methanol metabolism is the oxidation of methanol to formaldehyde by alcohol oxidase with molecular oxygen. There are two alcohol oxidase encoding genes in Pichia pastoris，AOX1 and AOX2. The majority of alcohol oxidase in wild-type yeast is encoded by AOX1，whose expression is strongly induced by methanol，with the AOX1 protein product accounting for over 30% of total cellular soluble protein. Many Pichia pastoris expression vectors utilize the AOX1 expression cassette for exogenous gene expression，and strains with functional AOX1 are defined as Mut?. AOX2 shares 97% sequence homology with AOX1 and compensates for the loss or dysfunction of AOX1，generating Mut? mutant strains with much weaker methanol oxidation capacity，reduced methanol metabolism efficiency and slower growth in methanol medium. It is difficult to predict whether Mut? or Mut? is more suitable as the host strain for the expression of a specific protein. Although Mut? yeast produces higher levels of transcribed mRNA，excessive mRNA does not always yield biologically active protein; by contrast，Mut? yeast grows slowly and generates less mRNA than Mut?，but may facilitate correct protein folding and the production of biologically active products. GS115 chemically competent cells are prepared using a specialized process，and the transformation efficiency determined with linearized pPIC9K plasmid (9. 3 kb，Amp? in E. coli) is greater than 10? cfu/μg DNA. Genotype: his4; Phenotype: His?，Mut?。