Targeting and retrofitting pre-existing libraries of transposon insertionswith FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment

A procedure is described that converts the pre-existing transposon insertio n libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and re trofitted with excision and amplification elements (FRT and oriV), by homol ogous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofit ted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitt ed transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions. To enhance the efficiency of crossing-in the FRT-oriV cassette, we transiently increased the copy num ber of our retrofitting plasmids using a temperature-sensitive TrfA-supplyi ng helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10Km(R) and three Tn10Cm(R) insertions. Subsequently, the FRT-oriV retrof itted insertions were crossed with each other in pairs (Km(R) x Cm-R), usin g P1 phage transductions. The resulting (CmFRT)-F-R-[28-65-kb]-Km(R)FRT str ains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled P-tet promoter. Induction of this tightly repressed pro moter by autoclaved chlortetracycline (cTc) resulted in the efficient excis ion and amplification of genomic fragments located between FRT sites, but o nly in productive strains, i.e, having two parallel FRTs. We have shown tha t genomic fragments of 28-, 40-. 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FR Ts in non-parallel orientation), to productive combination of parallel FRTs , because one of the FRT elements was flanked by two convergent loxP sites, and thus could be inverted by the Cre function delivered either by the P1 phage or by a specially constructed temperature-sensitive P-lac-cre plasmid . Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (pr epared without the conventional cloning and its artifacts) for refined sequ ence comparison among strains and species, and for further analysis of unch aracterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated l ike any other large plasmids but only in hosts that could supply the approp riate Rep function. Our original 'pop-out' method [Posfai et al. (1994) Nuc leic Acids Res. 22, 2392-2398] was already employed for sequencing of the E . coli genome [Blattner et al. (1997) Science 277, 1453-1474]. Moreover, th e Flp-mediated recombination between two FRT elements resulted in bacterial strains with large deletions (for parallel FRT orientations) or with large inversions (for inverted FRT orientations). (C) 1998 Elsevier Science B.V. All rights reserved.