• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Jun 25, 1994; 22(12): 2392–2398.
PMCID: PMC523700

In vivo excision and amplification of large segments of the Escherichia coli genome.

Abstract

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Greener A, Filutowicz MS, McEachern MJ, Helinski DR. N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. Mol Gen Genet. 1990 Oct;224(1):24–32. [PubMed]
  • Cox MM. The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4223–4227. [PMC free article] [PubMed]
  • Senecoff JF, Bruckner RC, Cox MM. The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7270–7274. [PMC free article] [PubMed]
  • Daniels DL, Plunkett G, 3rd, Burland V, Blattner FR. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science. 1992 Aug 7;257(5071):771–778. [PubMed]
  • Miller VL, Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. [PMC free article] [PubMed]
  • Bochner BR, Huang HC, Schieven GL, Ames BN. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. [PMC free article] [PubMed]
  • Dretzen G, Bellard M, Sassone-Corsi P, Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. [PubMed]
  • Koob M, Szybalski W. Preparing and using agarose microbeads. Methods Enzymol. 1992;216:13–20. [PubMed]
  • Koob M, Szybalski W. Cleaving yeast and Escherichia coli genomes at a single site. Science. 1990 Oct 12;250(4978):271–273. [PubMed]
  • Calos MP, Lebkowski JS, Botchan MR. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. [PMC free article] [PubMed]
  • Makino K, Shinagawa H, Amemura M, Nakata A. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J Mol Biol. 1986 Jul 5;190(1):37–44. [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • Heath JD, Perkins JD, Sharma B, Weinstock GM. NotI genomic cleavage map of Escherichia coli K-12 strain MG1655. J Bacteriol. 1992 Jan;174(2):558–567. [PMC free article] [PubMed]
  • Palchaudhuri S, Patel V, McFall E. DNA sequence of the D-serine deaminase activator gene dsdC. J Bacteriol. 1988 Jan;170(1):330–334. [PMC free article] [PubMed]
  • Tiedeman AA, DeMarini DJ, Parker J, Smith JM. DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12. J Bacteriol. 1990 Oct;172(10):6035–6041. [PMC free article] [PubMed]
  • Shafferman A, Kolter R, Stalker D, Helinski DR. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. [PubMed]
  • Szybalski W. From the double-helix to novel approaches to the sequencing of large genomes. Gene. 1993 Dec 15;135(1-2):279–290. [PubMed]
  • Nash HA. Integration and excision of bacteriophage lambda: the mechanism of conservation site specific recombination. Annu Rev Genet. 1981;15:143–167. [PubMed]
  • Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol. 1981 Aug 25;150(4):467–486. [PubMed]
  • Broach JR, Hicks JB. Replication and recombination functions associated with the yeast plasmid, 2 mu circle. Cell. 1980 Sep;21(2):501–508. [PubMed]
  • Golic KG, Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell. 1989 Nov 3;59(3):499–509. [PubMed]
  • O'Gorman S, Fox DT, Wahl GM. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991 Mar 15;251(4999):1351–1355. [PubMed]
  • Gronostajski RM, Sadowski PD. Determination of DNA sequences essential for FLP-mediated recombination by a novel method. J Biol Chem. 1985 Oct 5;260(22):12320–12327. [PubMed]
  • Beatty LG, Babineau-Clary D, Hogrefe C, Sadowski PD. FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction. J Mol Biol. 1986 Apr 20;188(4):529–544. [PubMed]
  • Kontomichalou P, Mitani M, Clowes RC. Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under either relaxed or stringent control. J Bacteriol. 1970 Oct;104(1):34–44. [PMC free article] [PubMed]
  • Kolter R, Inuzuka M, Helinski DR. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. [PubMed]
  • Stalker DM, Kolter R, Helinski DR. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol. 1982 Oct 15;161(1):33–43. [PubMed]
  • Metcalf WW, Jiang W, Wanner BL. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene. 1994 Jan 28;138(1-2):1–7. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...