High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation.
A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.
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
Hatakeyama
T
T
D015925:Cryopreservation; D003470:Culture Media; D003543:Cysteamine / Q000494:pharmacology; D004271:DNA, Fungal / Q000235:genetics*; D004229:Dithiothreitol / Q000494:pharmacology*; D018274:Electroporation; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010957:Plasmids; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D014170:Transformation, Genetic
D003470:Culture Media; D004271:DNA, Fungal; D013438:Sulfhydryl Compounds; D003543:Cysteamine; D004229:Dithiothreitol