Electroporation of Yeast (Pichia)

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Kingfisher's picture
Electroporation of Yeast (Pichia)

I am introducing genes for expression in yeast (KM71H strain) by electroporation using a pPICZa vector .  I have been getting variable results.  I typically recover transformed cells, but am trying to increase the efficiency to select on higher levels of antibiotic (supposedly higher copy number will grow on higher levels of antibiotic).  Any suggestions for pichia electroporation?  Any good websites or references for methods of cell preparation and/or actual EP conditions?  Thanks.

Monu's picture
Electroporation is a

Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.
Visit these web pages, may be of some help to you. http://www.thermo.com/com/cda/landingpage/0,10255,700,00.html
Electroporation Protocols: http://www.protocol-online.org/prot/Molecular_Biology/Transformation/Electroporation/index.html

varsha's picture

I have never worked with yeast myself. This paper may have some of the information you are loking for. http://www.find-health-articles.com/rec_pub_14740498-high-efficiency-transformation-electroporation-pichia-pastoris.htm
Good luck!

Ivan Delgado
Ivan Delgado's picture

As someone that has cracked open mammalian cells (tissue and cultured), plant cells, bacteria, yeast, etc, I can attest to the fact that it can be quite tricky to get through the outer membrane of yeast. In addition Pichia is one of those organisms that happens to get used mostly in industry, so protocols for its manipulation are limited at best. The fact that Pichia's genome was just sequenced last month (Nature Biotechnol, June 2009) emphasizes this fact. 
The standard way of electroporating Pichia is to follow the Invitrogen protocol, which is copied pretty much verbatim here. There are many other protocols available, but by far the most important consideration is the fact that you should use fresh Pichia competent cells. If you are trying to store your competent cells and then thawing them before use, then very likely the variation you are seeing in your experiments is due to the fact that Pichia do not store well. Prepare them fresh every time you need them.
Good luck