X33 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，store at -20 ℃ for standby after use，thaw Carrier DNA on ice before next use，and re-denature Carrier DNA after 3-5 repeated freeze-thaw cycles; 2. Transforming high-concentration plasmids can correspondingly reduce the final bacterial amount for plating; 3. The dosage of plasmids can be increased when simultaneously transforming 2-3 kinds of plasmids，and the total volume of plasmids shall not exceed 1/10 of the competent cell volume; 4. The suitable growth temperature of Pichia pastoris is 28 ℃ to 30 ℃，and temperature above 32 ℃ is harmful to protein expression and may lead to cell death. X-33 is a non-genetically modified wild-type Pichia pastoris strain applied for recombinant protein expression; when transformed with Pichia pastoris expression plasmids without His selection markers including pPICZA，B，C and pPICZαA，B，C，positive transformants can be screened by Zeocin or bleomycin hydrochloride，while recombinant positive colonies cannot be screened using auxotrophic medium，and X-33 presents the Mut+ phenotype. As a methylotrophic yeast，Pichia pastoris can utilize methanol as the sole carbon source and contains two alcohol oxidase-encoding genes，AOX1 and AOX2; strains with functional AOX1 are classified as Mut+ type，and AOX2，sharing 97% homology with AOX1，will function when AOX1 is deleted or loses its activity，generating Mut s mutant strains with weakened methanol metabolic capacity and slow growth rate in methanol medium. X-33 chemically competent cells are prepared via a special process，and the transformation efficiency verified with linearized pPICZA plasmid (3. 3 kb，Zeocin R expressed in E. coli and Pichia pastoris) is over 10 4 cfu/μg DNA，with the genotype of Wild type and phenotype of Mut+.