A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells:: Application for DNA fragment transfer between chromosomes and plasmid transformation
A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells:: Application for DNA fragment transfer between chromosomes and plasmid transformation
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DOI：
10.1016/j.mimet.2005.06.001
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发表时间：
2006-03-01
影响因子：
2.2
通讯作者：
Schweizer, HP
中科院分区：
文献类型：
--
作者：
Choi, KH;Kumar, A;Schweizer, HP
描述了一种基于快速微量离心机的方法，用于制备铜绿假单胞菌电感受态细胞，其转化效率比现有程序提高了 10,000 倍。这种效率的提高现在使得所有需要 DNA 转移的应用都可以使用转化。其中包括在铜绿假单胞菌菌株之间转移标记有抗生素抗性基因的染色体突变，这解决了该细菌没有有效且可靠的转导程序的难题。毫不奇怪，该方法还允许使用复制质粒进行非常有效的转化，转化效率范围为每微克 DNA 10(7) 至 > 10(11) 个转化子。最后，该方法的效率高达每微克 DNA > 10(3) 个转化体，在大多数情况下取代了用于基因替换、位点特异性基因整合和转座子诱变实验中的非复制质粒转移的接合。 (C) 2005 Elsevier B.V. 保留所有权利。
A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to > 10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to > 10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments. (C) 2005 Elsevier B.V. All rights reserved.