I have digested my plasmid with Stu1 as recommended for pAO815 plasmid to transform GS115 strain of Pichia Pastoris. My question is how to screen these transformants?
Either transformants will grow on histidine deficient medium (for example RDB). The restriction site of Stu1 exactly cut His4 of the plasmid into almost two equal sites ?
or antibiotics Screening should be prefered ?
you need to make selection first, test transformed plasmid stability then use the gel electrophoresis for excluding re-annealed plasmids
You have to select with -HIS first since you will kill alot of cells if you use antibiotic selection first (also has a higher rate of false positive for antibiotic selection).
you need to make selection first, test transformed plasmid stability then use the gel electrophoresis for excluding re-annealed plasmids
This Picchia strain has a defect His-gene. After transformation your plasmid is (hopefully) inserted into the Piccha genome in this His-gene creating two His-genes, one defect and one functional. Thus the selection is done on -His media.
After that I always picked clones and did a western blot (which we called Yeastern blot) to confirm Insertion of the plasmid and expression of the target protein. Most of these clones were positive. Thus if you have antibodies it is an easy way to confirm positive clones. An alternative could be a small scale expression and then test activity and/or run a protein gel.
When I did this I am rather sure we transformed Picchia with circular plasmids (not cut), it would give a higher transformation efficiency. Due to the presence of a His-gene in both the Picchia genome and the plasmid it is still possible for the plasmid to be taken up into the genome.
Yes pichia strain is -his4.
Sir Anders O Magnusson you means that it is not necessary to linearise plasmid before transformation into Pichia. Last time i did transformation with circular plasmid (not cut) but there was no growth on Regeneration dextrose medium (RDB) which is histidine deficient one.
Transformation Parameters were
(1) quantity of plasmid used 100ng/ul (introgen manual recommended 5-10 ug of plasmid).
(2) 80 ul of cells having od 1.5 (cells viability were checked on YPD plates as there was growth).
what will be the possible causes of no growth ?
Hanna Alalam which medium will be the most suitable medium for transformants selection by using pAO815 plasmid and with GS115 strain of pichia.
Khaled Khleifat sir Top10 F' ecoli cells are successfully transformed with same plasmid. transformants were selected on LB- AMp plates.
@ Matiullah Khan , I suggest you to perform colony PCR with Plasmid and your insert primers. More fine confirmation is the sequencing of your colony PCR product. I hope it will work the best.
Good Luck
Matiullah Khan Sorry my memory played me wrong. I also linearized the plasmid before transformation. Did transformation by electroporation into electro competent Picchia strain SMD1168.
Matiullah Khan the reason you need to linearize the plasmid before transformation is that you need to stimulate recombination with the chromosome. You are generating two copies of the his gene on the chromosome with your expression cassette in between. You can screen by expression as Anders O Magnusson has pointed out, and you can also screen by PCR but you need to be careful in your selection of primers to ensure you really are confirming the product of interest.
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