A modular system for Clostridium shuttle plasmids.

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.