RP4-based plasmids for conjugation between Escherichia coli and members of the vibrionaceae

The Vibrionaceae family includes both important pathogens and useful model organisms in the study of processes ranging from the regulation of light production by quorum sensing to the establishment of mutualistic animal-bacteria associations. The self-transmissible broad host-range IncPα plasmid RP4 provides the means for genetic manipulation of many diverse bacteria, including members of the Vibrionaceae. Derivatives of RP4 facilitate the conjugal transfer of vectors engineered using Escherichia coli (E. coli) as a host into other bacterial species of interest. The new RP4-based constructs described in the chapter are at least as efficient as existing tools for mediating conjugal transfer between E. coli and members of the Vibrionaceae. The tools and methods described in the chapter are also useful for mediating conjugation between E. coli and many other gram-negative bacteria. Helper plasmid pEVS 104 may be particularly advantageous for recipients, such as the Enterobacteriaceae, that replicate ColE1- based vectors or support transposition of IS903, Mu, Tn7, or E. coli IS elements.