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

We have constructed an R6K-based suicide vector that permits: the random in sertion of a mini-transposon named NKBOR into the chromosome of Gram-negati ve bacteria and the subsequent rapid cloning of sequences flanking the inse rtion 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 propagate d by replication in an E. coli strain containing the R6K replicase pi prote in. Alternatively the mini-transposon can be replicated in a pSC101 derivat ive that is thermosensitive for its replication so that the mini transposon acts as a suicide plasmid at nonpermissive temperatures. Efficient NKBOR t ransposition is ensured by expression of an adjacent transposase gene and h as been demonstrated in E. coli, Klebsiella pneumoniae, and Erwinia carotov ora. Sequences flanking the insertion sites in these strains can be rapidly recovered and identified in E. coli strains expressing the R6K pi protein. (C) 2001 Editions scientifiques et medicales Elsevier SAS.