质粒DNA对乳糖发酵短杆菌原生质体的高频转化
High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.
作者信息
Santamaria R I, Gil J A, Martin J F
出版信息
J Bacteriol. 1985 Apr;162(1):463-7. doi: 10.1128/jb.162.1.463-467.1985.
Abstract
An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.
摘要
已开发出一种高效的聚乙二醇辅助方法，用于使用质粒载体转化乳酸发酵短杆菌原生质体。选择了两个小质粒，pUL330（5.2千碱基）和pUL340（5.8千碱基）作为载体，它们都含有来自转座子Tn5的卡那霉素抗性基因和乳酸发酵短杆菌天然质粒pBL1的复制起点。质粒的超螺旋形式产生的转化频率比线性形式高100倍。在1毫升转化缓冲液中加入10纳克DNA可实现最佳转化频率。更高浓度的质粒DNA导致每微克DNA的转化频率降低。使用25%至35%的聚乙二醇6000可获得最佳转化效果。在最佳条件下，每微克DNA可获得10⁶个转化体。
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