Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts
Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts
复制标题
伏氏甲烷球菌原生质体的自然和电穿孔介导转化
DOI：
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发表时间：
1994
影响因子：
4.4
通讯作者：
G. Sprott
中科院分区：
文献类型：
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作者：
G B Patel;John H. E. Nash;Brian J. Agnew;G. Sprott
由于缺乏高效的转化系统，严重阻碍了古细菌界产甲烷菌的遗传研究。通过使用甲烷球菌的原生质体和整合载体，Mip 1，以前显示赋予嘌呤霉素抗性，我们获得了自然转化频率约80倍高（705转化体/μg转化DNA）比报道的全细胞。电转化M.具有共价闭合环状Mip 1 DNA的原生质体是可能的，但在较低的频率下，约。177个转化体/μg载体DNA。然而，通过用线性化DNA电穿孔原生质体，实现了比用全细胞天然转化的频率提高380倍（3，417个转化体/μg DNA）。这种使用原生质体的一般方法应该允许转化其他产甲烷菌，特别是那些由于在低渗溶液中倾向于裂解而可能温和地转化为原生质体的产甲烷菌。
The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.