在难培养细菌中的高通量克隆与表达

High-throughput cloning and expression in recalcitrant bacteria.

作者信息

Geertsma Eric R, Poolman Bert

机构信息

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.

出版信息

Nat Methods. 2007 Sep;4(9):705-7. doi: 10.1038/nmeth1073. Epub 2007 Jul 22.

DOI:10.1038/nmeth1073

PMID:17643108

Abstract

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.

摘要

我们开发了一种用于在不太适合常规DNA操作的细菌中进行高通量克隆的通用方法。该方法包括在中间大肠杆菌载体中进行不依赖连接的克隆，通过载体骨架交换（VBEx）将其快速转化为适合高效转化的特定生物体的质粒。我们展示了乳酸乳球菌的VBEx原理证明，但该方法可适用于所有有可用质粒的生物体。