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Construction of delivery vectors for miniTn7BB

In order to propagate the miniTn7BB-Gm minitransposon and to facilitate further genetic manipulation and transfer to the target strains for transposition, we decided to construct two different delivery plasmids, based on the vectors pUC18Sfi (Herrero et al., 1990) and pUC18R6KT (Choi et al., 2005). pUC18Sfi is a pUC18-derived cloning vector harboring an ampicillin resistance marker, a high copy number mutant pMB1 replication origin and the pUC18 multi-cloning site flanked by two SfiI restriction sites (Figure 2,A). Because of its high copy number and its ability to replicate in any E. coli strain and in other enterobacteria, pUC18Sfi is suitable for maintenance of miniTn7 transposon derivatives, and for simple plasmid preparation and genetic manipulation. pUC18Sfi can be used to deliver transposons by transformation in bacteria in which it does not replicate (i.e., non-enteric bacteria), but not in the enterics. pUC18R6KT is also a pUC18-derived ampicillin resistant vector, but it harbors the low- to medium-copy number R6K replication origin, which is dependent on the presence of the replication protein π, and is only replicative in a small subset of E. coli strains engineered to express π (typically from a specialized λ prophage named λ-pir). Genetic manipulation of pUC18R6KT-derived plasmids is more cumbersome because of its lower copy number and the requirement of specific E. coli strains, but their major advantage over pUC18Sfi-based plasmids is that they can be used for transposon delivery in E. coli and other enterobacteria. In addition, the presence of mobilization functions enables conjugative transfer of pUC18R6KT and its derivatives to a wide range of recipient bacteria (Figure 2,B).

Figure 2. Structure of the pUC18Sfi (left) and pUC18R6K-T (right) plasmids.

In order to construct the pUC18Sfi-miniTn7BB-Gm delivery plasmid, the miniTn7BB-Gm transposon was cleaved from the commercial plasmid pMA (Mr. Gene) with SfiI and ligated to SfiI-digested pUC18Sfi. This strategy removes all multi-cloning restriction sites from pUC18Sfi, except for the flanking SfiI, thus guaranteeing that the unique sites in the transposon are not duplicated. The remove of the multi-cloning restriction sites was verify by analytic digestions. In order to construct the pUC18R6KT-miniTn7BB-Gm delivery plasmid, the pUC18R6KT backbone was reverse PCR-amplified with primers bearing SfiI sites using as template the pTNS2 plasmid (Choi et al., 2005) and the PCR product was digested with SfiI and ligated to SfiI-cleaved miniTn7BB-Gm. Again, this strategy removes all polylinker restriction sites, leaving the unique sites in the transposon as such. In both cases, the orientation of the miniTn7BB-Gm insertion was determined by digestion.