Hello,
I am attempting to co-express two different genes in Pichia pastoris X-33. My colleague successfully achieved this by transforming two different vectors simultaneously. Essentially, he added two distinct plasmids to the competent cells and performed electroporation. However, I am struggling to replicate this efficiently without resorting to multiple attempts and relying on luck. Do you have any suggestions on how to enhance the efficiency of this process?
One idea was to transform one gene and then the other one, with each vector containing a different antibiotic resistance. However, this strain is known to use only Zeocin (correct me if I am wrong).
Another obvious answer would be to create a construct with two different genes. However, in my work, I need to use any different gene combination. So, this would add too much extra work.
Thank you, and I hope you're all having a lovely New Year.
I am not using Pichia system for expressing multiple proteins but my suggestion to you is not to use the same selective marker for each plasmid (if both plasmids have Zeocin it is impossible to distinguish just by selection between a single or a double transformant). X-33 is a wild type strain therefore you can surely use also other markers like KanR (G418 resistance) or Hph (hygromycin B resistance). Using plates containing both drugs should help you to select double transformants. Hope this helps.
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