Takahashi W, Yamagata H, Yamaguchi K, Tsukagoshi N, Udaka S
J Bacteriol. 1983 Dec;156(3):1130-4. doi: 10.1128/jb.156.3.1130-1134.1983.
A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.
已开发出一种将质粒DNA导入短芽孢杆菌47（一种蛋白质分泌细菌）的方法。用碱性pH值的50 mM Tris - 盐酸缓冲液处理短芽孢杆菌47细胞对于诱导DNA摄取能力是有效的。在聚乙二醇存在的情况下，当向10⁹个经Tris处理的细胞中加入1微克质粒DNA时，经Tris处理的细胞以10⁻⁴的频率（每存活细胞中的转化体数）摄取质粒DNA。Tris - 盐酸缓冲液的pH值以及聚乙二醇的浓度和分子量影响转化频率。短芽孢杆菌47细胞的生长阶段对频率有强烈影响。通过这种方法已将两种质粒pHW1和pUB110导入短芽孢杆菌47。讨论了用Tris - 盐酸缓冲液处理短芽孢杆菌47细胞与去除细胞壁外层两个蛋白质层相关的DNA摄取能力诱导机制。
