Targeting and retrofitting pre-existing libraries of transposon insertions with 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 insertion 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 retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted 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 number of our retrofitting plasmids using a temperature-sensitive TrfA-supplying helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10KmR and three Tn10CmR insertions. Subsequently, the FRT-oriV retrofitted insertions were crossed with each other in pairs (KmRxCmR), using P1 phage transductions. The resulting CmRFRT-[28-65-kb]-KmRFRT strains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled Ptet promoter. Induction of this tightly repressed promoter by autoclaved chlortetracycline (cTc) resulted in the efficient excision and amplification of genomic fragments located between FRT sites, but only in productive strains, i.e. having two parallel FRTs. We have shown that genomic fragments of 28-, 40-, 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FRTs 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 Plac-cre plasmid. Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (prepared without the conventional cloning and its artifacts) for refined sequence comparison among strains and species, and for further analysis of uncharacterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated like any other large plasmids but only in hosts that could supply the appropriate Rep function. Our original 'pop-out' method [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398] was already employed for sequencing of the E. coli genome [Blattner et al. (1997) Science 277, 1453-1462]. Moreover, the 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).
Wild
J
J
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706, USA.
Sektas
M
M
Hradecná
Z
Z
Szybalski
W
W
D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D016678:Genome; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin; D014764:Viral Proteins
D004251:DNA Transposable Elements; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases