Intact Cell Transformation ofSaccharomyces cerevisiaeby Polyethylene Glycol
Intact Cell Transformation ofSaccharomyces cerevisiaeby Polyethylene Glycol
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聚乙二醇对酿酒酵母的完整细胞转化
DOI：
10.1080/00021369.1985.10866817
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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
利用完整细胞代替原生质体转化酵母的新方法近年来得到了发展。2)这种转化方法基于用碱阳离子（如醋酸锂或硫醇化合物）处理完整的细胞。与目前的原生质体方法相比，它具有几个优点；例如，由于可以避免酵母菌细胞壁的溶酶消化，因此不需要琼脂再生，并且转化菌落的出现明显更快（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转化。在本文中，我们为这一观察提供了证据。转化过程主要基于Ito等人报道的方法[2]。将酿酒酵母菌AH22 （a leu2-3 iu2 -12 his4-519 canl）、YNN27 （c his3-532 trpl gall）和D13-1A （a his3-532 trpl gall）在1ml YEPD（1.0%酵母提取物、2.0%多肽、2.0%葡萄糖、pH 5.3）中预培养过夜。20ml的yepd含有0.4 ml的预培养细胞悬液，在30℃下摇晃孵育，直到细胞生长到log晚期。1000xg离心5min收获细胞，用TEbuffer （10mMTris-HCl buffer, pH 7.5，含1mMEDTA）洗涤一次。然后将细胞以2 × 108个/ml的浓度悬浮在同一缓冲液中。然后将500 ^ 1的含0.2 m醋酸锂的TEbuffer （Ito et al. u2的醋酸锂法）或单独的TEbuffer（聚乙二醇法）加入500 [A的细胞悬液中。
Anewyeast transformation methodusing intact cells instead ofprotoplasts has recently beendeveloped. 1> 2) This transformation methodis based onthe treatment of intact cells with alkali cations such as lithium acetate or thiol compounds. It has several advantages compared with the current protoplast method3); for example, since lytic en-zyme digestion of yeast cell walls can be avoided, re-generation agar is not necessary andtransformant colonies appear significantly faster (2~ 4days). In the course of efforts to develop newyeast host vector systems wehave found that intact cells treated with only polyethylene glycol (PEG) can be transformed. In this paper wepresent 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 0.4 ml 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 TEbuffer (10mMTris-HCl buffer, pH 7.5, containing 1mMEDTA). Thenthe cells were suspended at a concentration of 2 x 108 cells/ml in the same buffer. Five hundred^ 1 of TEbuffer containing 0.2 m lithium acetate (lithium acetate methodof Ito et al. u2)) or the samevolumeof TEbuffer alone (polyethylene glycol method) was then added to 500 [A of the cell suspension.