Intact Cell Transformation of Saccharomyces cerevisiae by Polyethylene Glycol
Intact Cell Transformation of Saccharomyces cerevisiae by Polyethylene Glycol
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聚乙二醇对酿酒酵母的完整细胞转化
DOI：
10.1271/bbb1961.49.869
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
N. Gunge
中科院分区：
文献类型：
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作者：
M. Yamakawa;F. Hishinuma;N. Gunge
不需要代琼脂，并且菌落出现明显更快（2 ~4天）。在努力开发新的酵母宿主载体系统的过程中，我们发现仅用聚乙二醇（PEG）处理的完整细胞可以被转化。在本文中，我们提出的证据，这一观察。转化过程主要基于Ito等人报道的方法。2）酿酒酵母菌株AH 22（a leu2-3 leu2 -12 his4-519 canl），YNN27（α trpl ura3 gall）和D13-1A（his 3 -532 trpl gall）在1 ml YEPD（1.0%酵母提取物，2.0%多肽，2.0%葡萄糖，pH5.3）中预培养过夜。将含有0.4ml预培养细胞悬浮液的20 ml YEH）在30°C下振荡孵育，直至细胞生长达到对数晚期。然后通过以1000 × g离心5分钟收获细胞，并用TE缓冲液（10 mM Tris-HCl缓冲液，pH 7.5，含有1 mMEDTA）洗涤一次。然后将500 μ L含有0.2M乙酸锂的TE缓冲液（Ito等人的乙酸锂方法）或相同体积的单独的TE缓冲液（聚乙二醇方法）加入到500 μ L细胞悬浮液中。在30°C下振荡孵育1小时后，
generation agar is not necessary and transformant colonies appear significantly faster (2 ~4 days). In the course of efforts to develop new yeast host vector systems we have found that intact cells treated with only polyethylene glycol (PEG) can be transformed. In this paper we present evidence for this observation. The transformation procedure is principally based on the method reported by Ito et al.lt2) The Saccharomyces cerevisiae strains, AH22(a leu2-3 Ieu2-12 his4-519 canl), YNN27 (oc trpl ura3 gall) and D13-1A (a his3-532 trpl gall), were precultured overnight in 1 ml of YEPD(1.0% yeast extract, 2.0% polypepton, 2.0% glucose, pH 5.3). Twenty ml of YEPDcontaining 0.4ml of a precultured cell suspension was incubated at 30°C with shaking until cell growth reached the late log phase. Cells were then harvested by centrifugation at 1000xg for 5min and washed once with TE buffer (10mM Tris-HCl buffer, pH 7.5, containing 1mMEDTA).Then the cells were suspended at a concentration of 2 x 108 cells/ml in the same buffer. Five hundred ^1 of TE buffer containing 0.2m lithium acetate (lithium acetate method of Ito et al.u2)) or the same volume of TEbuffer alone (polyethylene glycol method) was then added to 500[A of the cell suspension. After 1hr incubation at 30°C with shaking, a 100}A