Cryopreservation of competent intact yeast cells for efficient electroporation.
We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long-term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non-permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10 degrees C/min and a rapid thawing rate of 200 degrees C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non-electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at -80 degrees C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long-term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation.
Suga
M
M
Department of Materials and Biosystem Engineering, Toyama University, 3190 Gofuku Toyama-City, Toyama 930-8555, Japan. suga@eng.toyama-u.ac.jp
Isobe
M
M
Hatakeyama
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D015925:Cryopreservation; D003470:Culture Media; D018274:Electroporation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D013012:Sorbitol / Q000494:pharmacology; D014170:Transformation, Genetic
D003470:Culture Media; D013012:Sorbitol; D000255:Adenosine Triphosphate