Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides Free

The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the BglII restriction enzyme, ligated to the 2.7 BglII fragment of transposon Tn10, which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides.