Recently, I got problems about Pichia expression. My target gene was inserted into pPIC9K vector via EcoRI and NotI restriction sites. And then the SacI digested construct was transformed into GS115 by electroporation. More than 200 transformants were obtained from the MD plate, and whcih was collected and subsequently plated on YPD plated with G418 gradient(0.5mg/ml to 3mg/ml). Only a few colony formed on the high G418 concentration plate, and then the colony was streaked on new G418 plate for isolation single clone. Then the genome DNA was extracted and PCR was performed.
PCR employed 5'AOX1 and 3'AOX1 primers gave 2.1Kbp bond, indicated the AOX1 gene from Pichia was still remained. however, PCR with my target gene speical primers, no bond was obtained. And subsequent western blot analysis shown that no target protein was expressed by induced expression.
Help!!!Where am I going wrong?
Hi,
- Did you confirmed insertion of your target gene into the vector before yeast transformation ? How about the ScaI enzyme not cutting within your gene of interest?
- According to Invitrogen, linearizing with ScaI generates Mut+ transformants, so your screening with 5'-AOX and 3'-AOX should give two bands: one corresponding to the insert and the AOX gene (the one you got). Therefore your PCR indicates no insert what means your had false positives transformants.
It has been reported that about 10 % to 20 % of the transformants are the result of a gene conversion event between the his4 gene of the transforming vector and the mutant his4 locus in the Pichia genome. In this conversion events, only the marker gene from the vector integrates into the mutant host locus without other vector sequences. Accordingly, make sure of the quality of your expression vector to be used for yeast transformation. However, it is also known that ''screening on geniticin is sensitive to the density of cells''
Hope this help you. Good luck.
If you do not have more "single-copy" transformants on plates with low concentration of the drug (0.5 and 1 g/l), it might be a case when all your transformants are false positive. I would suggest you to check out by PCR the transformants you already checked together with less resistant transformants using the primers for your gene and some vector specific primers, especially for those that interact with an upstream of the AOX1 promoter region. I'd suggest you not to waste your time with extraction of DNA from transformats but a colony PCR approach (boil a dense suspension of you culture in small volume (100 - 300 ul) of water and use 10-25 ul of the liquid (without cells) as DNA template. The circular plasmid is a good positive control for such PCRs.
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