NKBOR, a mini-Tn10-based transposon for random insertion in the chromosome of Gram-negative bacteria and the rapid recovery of sequences flanking the insertion sites in Escherichia coli.

We have constructed an R6K-based suicide vector that permits the random insertion of a mini-transposon named NKBOR into the chromosome of Gram-negative bacteria and the subsequent rapid cloning of sequences flanking the insertion site in Escherichia coli. This mini-transposon contains a conditional R6K plasmid origin of replication, a kanamycin resistance gene and unique restriction sites between the IS10 inverted repeats. NKBOR can be propagated by replication in an E. coli strain containing the R6K replicase pi protein. Alternatively the mini-transposon can be replicated in a pSC 101 derivative that is thermosensitive for its replication so that the mini-transposon acts as a suicide plasmid at nonpermissive temperatures. Efficient NKBOR transposition is ensured by expression of an adjacent transposase gene and has been demonstrated in E. coli, Klebsiella pneumoniae, and Erwinia carotovora. Sequences flanking the insertion sites in these strains can be rapidly recovered and identified in E. coli strains expressing the R6K pi protein.