I am trying to express my protein in Pichia pastoris X-33. I have cloned my gene in NotI and XbaI sites in pPICZalpha and after linearizing with PmeI and electroporation I plate on YPDS + Zeocin plates. After getting colonies, when I carry out PCR with AOX1 primers, I get two bands of size 2.2 kb (corresponding to native AOX1 gene) and 1.3 kb band (800 my gene size + 588 bp for vector) indicating colonies are Mut+ but when I plate them on MM and MD plate, I get slow growth on MM than MD indicating Muts phenotype and I am getting very weak expression. I am confused as to whether my clones are Mut+ or Muts? Can anyone help
Transformation of X-33 or GS115 with linearized constructs favor single crossover
recombination at the AOX1 locus. Most of the transformants should be Mut+;
however, with the presence of the AOX1 sequences in the plasmid, there is a
chance that recombination will occur in the 3´AOX1 region also, disrupting the
wild-type AOX1 gene and creating MutS transformants.
So i advise you to screen more clones (~100 clones) from higher concentration of Zeocin plates. It may solve you both doubling time & yield related problems
If you already screened many clones and still facing same problem, then the BEST & alternative strategy to improve yields by Multimerization of your gene castes..
At least you will find ~10-20 folds more specific yield then monomeric approach..
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