Heap John T, Pennington Oliver J, Cartman Stephen T, Minton Nigel P
BBSRC Sustainable BioEnergy Centre, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
J Microbiol Methods. 2009 Jul;78(1):79-85. doi: 10.1016/j.mimet.2009.05.004. Epub 2009 May 13.
Despite their medical and industrial importance, our basic understanding of the biology of the genus Clostridium is rudimentary in comparison to their aerobic counterparts in the genus Bacillus. A major contributing factor has been the comparative lack of sophistication in the gene tools available to the clostridial molecular biologist, which are immature, and in clear need of development. The transfer and maintenance of recombinant, replicative plasmids into various species of Clostridium has been reported, and several elements suitable as shuttle plasmid components are known. However, these components have to-date only been available in disparate plasmid contexts, and their use has not been broadly explored. Here we describe the specification, design and construction of a standardized modular system for Clostridium-Escherichia coli shuttle plasmids. Existing replicons and selectable markers were incorporated, along with a novel clostridial replicon. The properties of these components were compared, and the data allow researchers to identify combinations of components potentially suitable for particular hosts and applications. The system has been extensively tested in our laboratory, where it is utilized in all ongoing recombinant work. We propose that adoption of this modular system as a standard would be of substantial benefit to the Clostridium research community, whom we invite to use and contribute to the system.
尽管梭菌属在医学和工业上具有重要意义，但与芽孢杆菌属中的需氧菌相比，我们对梭菌属生物学的基本理解还很初步。一个主要因素是梭菌分子生物学家可用的基因工具相对不够成熟，明显需要进一步开发。已有将重组复制质粒转入各种梭菌属物种以及一些适合作为穿梭质粒组分的元件的报道。然而，这些组分迄今为止仅存在于不同的质粒背景中，其应用尚未得到广泛探索。在此，我们描述了一种用于梭菌 - 大肠杆菌穿梭质粒的标准化模块化系统的规格、设计和构建。我们纳入了现有的复制子和选择标记，以及一种新型的梭菌复制子。我们比较了这些组分的特性，这些数据使研究人员能够识别可能适用于特定宿主和应用的组分组合。该系统已在我们实验室进行了广泛测试，并用于所有正在进行的重组工作中。我们建议采用这个模块化系统作为标准将对梭菌研究界大有裨益，我们邀请该领域的研究人员使用并为该系统做出贡献。
