Restriction digests of the clone give the following sizes (kb): EcoRI--13.0, 5.9; BamHI--12.0, 7.6; ClaI--17.0, 2.6; XhoI--19.6. E. coli S17-1 contains an integrated derivative of RP4 and allows conjugative transfer of the vector. Transconjugants are identified by resistance to tetracycline and carbenicillin. In some strains, carbenicillin resistance may depend on transcription originating outside the transposon. This can provide a means for direct selection of recombinants in which the Tn has inserted downstream from an active promoter. Chromosomal DNA fragments adjacent to the inserted lacZ gene can be cloned following digestion with HindIII, SalI, SmaI or XhoI and religation of the vector. Fragments adjacent to the transposase can be cloned following digestion with BamHI, ClaI, EcoRI or SacI and religation. It should also be possible to clone DNA flanking both sides of the transposon using BglII. Religated vector can be transformed and propagated in any suitable E. coli, such as E. coli MC1061. Integrating, "self-cloning" promoter probe vector. A mobilizable suicide plasmid that can be transferred to a broad range of Gram-negative bacteria. The order of the major features in the plasmid is: IR - BamHI - lacZ(ClaI, SacI) - EcoRI - ampR - pMB1 ori - tetA - tetR(SalI, SmaI) - SmaI (2 sites) - tnp(HindIII, XhoI) - IR - BamHI - SalI - mob - cmlR(EcoRI) - ClaI - HindIII - gentamicinR. |
