The efficient strategy of plasmid rescue from Tn5 mutants derived from Bradyrhizobium japonicum Is-1, based on whole genome sequence information of strain USDA110

We previously reported that Tn5 mutants derived from Bradyrhizobium japonicum Is-1 were isolated. In this report, Tn5-encoded kanamycin resistance cassette and its flanking sequence were concurrently cloned into cosmid vector from Tn5 mutants. In general, the genomic southern blot analysis is often performed to select appropriate restriction enzyme before performing the plasmid rescue from cosmid clone. Therefore plasmid rescue experiment consumes the time, especially when more than one Tn5 mutant was obtained. To improve this situation, tentative restriction map of strain Is-1 was constructed based on whole genome sequence information of strain USDA110. Plasmid rescue was performed with restriction enzymes selected according to this map and these enzymes were appropriate for plasmid rescue. This strategy is thought to be effective for saving the time of plasmid rescue in B. japonicum strains other than USDA1 10.