The objective of this proposal is to develop a system in which an investigator who has found that a cloned gene is expressed poorly in E. coli can rapidly convert this already existing plasmid into a new plasmid capable of replication in all gram negative bacteria. This conversion relies on homologous recombination in vivo, and therefore, does not require restriction enzymes, subcloning, or DNA preparations. The advantage to screening other bacteria besides E. coli for protein production is based on the assumption that since many unrelated species of hosts can be analyzed simultaneously, one or more are likely to provide a more suitable environment for a particular protein. This approach is considerably more rapid and economical than redesigning vectors or changing expression systems when E. coli does not overproduce the foreign protein of interest.
