KM71H Chemically Competent Cell

1.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. Store it at -20 ℃ for standby after use. Thaw Carrier DNA on ice before next use. Re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles; 2. The pPICZA plasmid needs to integrate into the Pichia pastoris genome and must be linearized by single enzyme digestion before transformation，with SalI commonly used for linearization; 3. Single-copy insertion of the Zeocin resistance gene into the Pichia pastoris genome can be screened at 100 μg/mL Zeocin/Bleomycin Hydrochloride. Multi-copy integration can improve the resistance level of Pichia pastoris to Zeocin/Bleomycin Hydrochloride，and the concentration can be increased according to experimental needs to screen strains with high-copy protein expression cassettes; 4. The optimal growth temperature of Pichia pastoris KM71H is 28℃ to 30℃，and temperatures above 31℃ will affect yeast growth. KM71H is a Pichia pastoris strain dedicated to recombinant protein expression. KM71H can express the HIS4 gene and cannot be used to screen recombinant positive colonies via auxotrophic medium; Pichia pastoris expression plasmids carrying the Zeocin resistance gene such as pPICZ A，B，C and pPICZα A，B，C can be screened with 100 μg/mL Zeocin or bleomycin hydrochloride. The phenotype of KM71H is Mut?，Arg?. The parent strain of KM71H has a mutation in the argininosuccinate lyase gene (arg4)，and insertion of the wild-type ARG4 gene at the AOX1 locus endows KM71H with the Arg? phenotype; meanwhile，the insertion disrupts the AOX1 gene，resulting in a Mut? phenotype. There are two genes encoding alcohol oxidase in Pichia pastoris，namely AOX1 and AOX2. Most alcohol oxidase in wild-type Pichia pastoris is encoded by the AOX1 gene，whose expression can be strongly induced by methanol，and the AOX1 protein product can account for more than 30% of the cellular soluble protein. Many Pichia pastoris expression plasmids utilize the AOX1 expression cassette for exogenous gene expression，and strains with functional AOX1 are defined as Mut? type. AOX2 shares 97% sequence homology with AOX1 and becomes functional when AOX1 is deleted or non-functional，generating Mut? mutant strains. The methanol oxidation capacity of AOX2 is much weaker than that of AOX1，leading to impaired methanol metabolism and slow growth in methanol medium. It is difficult to predict whether Mut? or Mut? is more suitable as the host for the expression of a specific protein. Mut? yeast yields more transcribed mRNA，yet excessive mRNA does not necessarily produce biologically active proteins; although Mut? yeast grows slowly and produces less mRNA than Mut?，it tends to facilitate correct protein conformation and the formation of biologically active proteins. KM71H chemically competent cells are prepared by a proprietary process and can be stored for three months at -80°C. The transformation efficiency is over 10? cfu/μg DNA as determined using linearized pPICZ A plasmid (3. 3 kb，Zeocin? in E. coli / Pichia pastoris). Genotype: aox1::ARG4，arg4; Phenotype: Mut?，Arg?.