Zeocine resistence, pichia pastoris - (Aug/24/2010 )

Hi
I try to express some protein in Pichia pastoris.
And I have the problem: when I carry out electroporation and spread yeast on YPD plates with zeocine ( 100ug/ml, 500ug/ml ), yeast grow only one day and the plate is full of yeast no colonies just coating. I tried to spread P.pastoris without vector contained zeocine resistence ( pPICZalpha)like negativ control. And the plate is coating again. The same I have when I spread E.coli nova blue or TOP10 on the LB low salt medium plates containing zeocine. I changed zeocine,
I controled preparing of medium and the result is same.
I do not know what I do now.
Could you help me somebody?
Thank you very much

Have you titrated the zeocin out to see a kill curve? Is your zeocin off?

bob1 on Tue Aug 24 22:25:57 2010 said:

I used new zeocine. I had not titrated zeocine. Could you advise me how I can do it?
Do you think that can be problem in used media?

Hello,

I hope is not to late to give you some advise. Zeocin can be a nightmare because is highly sensitive to salt in medium. For me you main problem seem to be the agar, let me explain: Beleive it or not yeast extract, agar, peptone and even glucose can inhibit zeocin. We had several problems when new glucose arrived to the lab. Other day we had troubles because the yeast extract. Because your problem seem to be with LB and YPD-sorbitol I will think first in agar, the best is to use ultra pure agar. Standar agar for medium is high in salt and does not work for zeocin.

Other thing you can try is to down yeast extract concentration to 0.5%

Last try, even you are getting plenty of colonies after you transformation try to select some positive transformants in liquid medium. To do that you need first to do liquid medium with zeocin (less yeast extract) and tested by growing your wild strain, if works in liquid medium you can pick 10 colonies and selected in 2 ml of liquid medium.

Hope I can help