Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium
A transposon Tn10 vehicle was developed using a self transmissible (Tra+) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 105 folds higher than the spontaneous TetR mutants. Reversion frequency of these mutants was less than 10−8 indicating adequate stability of Tn10 mutations.
Key wordstransposon Tn10 vector insertion mutagenesis Rhizobium
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