Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event

Abstract

Background: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage lambda red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step.

Results: We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion.

Conclusion: This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct.

Figure 1

Transposition vector. The vector pGRG25 contains a multiple cloning site (MCS) with unique restriction sites for AvrII, NotI, PacI, and XhoI, flanked by the terminal repeats of Tn7. Cloning of the gene of interest into these sites, in between the Tn7 arms, will result in a plasmid that can be used to insert the transgene into the chromosome. araC is the regulator of the arabinose-inducible promoter P_BAD (though in all experiments presented, leaky expression was sufficient). tnsABCD are the genes required for transposition of Tn7. rrnD is the transcription terminator. bla encodes β-lactamase which allows selection of ampicillin resistance when transforming the plasmid into bacteria. oriT is the RP4 conjugal transfer origin. pSC101 ori ts is the temperature sensitive origin of replication which functions at 32°C, but not at 42°C. Vector pGRG36 is identical except for the addition of a SmaI site to the MCS. The sequence of pGRG25 and pGRG36 can be accessed at GenBank (accession # DQ460223).

Figure 2

Protocol for use of transgene insertion plasmid. 1) Transform the plasmid into the strain of choice and select transformants on LB + ampicillin at 32°C. Introduction of the plasmid by conjugation is also an option. 2) Streak once to ensure that the cells are carrying the plasmid, then grow non-selectively in LB at 32°C. In all strains tested thus far, leaky expression of TnsABCD during this step has been sufficient to allow site-specific transposition into the attTn7, but 0.1% arabinose may be added to increase levels of expression of TnsABCD. 3) Plate cells at 42°C to block replication of the plasmid. 4) Streak once at 42°C to ensure loss of the plasmid, then verify insertions in the attachment site by PCR.