A BROAD-HOST-RANGE IN-VIVO POP-OUT AND AMPLIFICATION SYSTEM FOR GENERATING LARGE QUANTITIES OF 50 TO 100-KB GENOMIC FRAGMENTS FOR DIRECT DNA-SEQUENCING

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employe d the yeast Flp/FRT elements for excision and the plasmid R6K-based re plication machinery for DNA amplification, permits one to bypass conve ntional cloning [Posfai et al. (1994) Nucleic Acids Res. 22, 2392-2398 ]. To extend the applicability of such a system to many species, we de scribe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep ( TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructe d insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a shor t genomic sequence homologous to this position was cloned into the mul tiple cloning site (MCS) of the FRT/oriV insertion plasmid and then re combined into this position in the genome by RecA-mediated recombinati on. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or ph age transduction allow two neighboring FRT/oriV sites (less than 150 k b apart) to be brought into a single genome. In the present report, th e lacZ and phoB sites, which are 51 kb apart in the E. coli genome, we re used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the P-tet promoter/operator which is tightly regulated by the TetR repressor. Th e addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) exc ision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.