Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Feb;176(4):1188–1191. doi: 10.1128/jb.176.4.1188-1191.1994

Molecular cloning of bacterial DNA in vivo using a transposable R6K ori and a P1vir phage.

I Stojiljkovic 1, J Bozja 1, E Salaj-Smic 1
PMCID: PMC205173  PMID: 8106331

Abstract

A new method of cloning in vivo using the P1vir phage and transposon Tn5-rpsL oriR6K was developed. The method relies upon recircularization of transducing DNA containing a transposon insertion in a recombination-deficient strain of Escherichia coli K-12 and subsequent stable replication of the recircularized DNA. Using this method, we were able to clone in vivo the chromosomal region located between approximately 7.1 and 9.2 min on the E. coli K-12 map in a 95-kb plasmid.

Full text

PDF
1188

Images in this article

1189Image
on p.1189
1190Image
on p.1190
1190Image
on p.1190

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biek D. P., Cohen S. N. Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. J Bacteriol. 1986 Aug;167(2):594–603. doi: 10.1128/jb.167.2.594-603.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chaudhury A. M., Smith G. R. A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7850–7854. doi: 10.1073/pnas.81.24.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Conley E. C., Saunders V. A., Jackson V., Saunders J. R. Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA. Nucleic Acids Res. 1986 Nov 25;14(22):8919–8932. doi: 10.1093/nar/14.22.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Darzins A., Casadaban M. J. In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage. J Bacteriol. 1989 Jul;171(7):3917–3925. doi: 10.1128/jb.171.7.3917-3925.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fellay R., Krisch H. M., Prentki P., Frey J. Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria. Gene. 1989;76(2):215–226. doi: 10.1016/0378-1119(89)90162-5. [DOI] [PubMed] [Google Scholar]
  6. Furuichi T., Inouye M., Inouye S. Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome. J Bacteriol. 1985 Oct;164(1):270–275. doi: 10.1128/jb.164.1.270-275.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gowrishankar J., Pittard J. Construction from Mu d1 (lac Apr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of lambda ppheA-lac. J Bacteriol. 1982 Jun;150(3):1122–1129. doi: 10.1128/jb.150.3.1122-1129.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Groisman E. A., Casadaban M. J. Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons. J Bacteriol. 1987 Feb;169(2):687–693. doi: 10.1128/jb.169.2.687-693.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Groisman E. A., Casadaban M. J. Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J Bacteriol. 1986 Oct;168(1):357–364. doi: 10.1128/jb.168.1.357-364.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groisman E. A., Castilho B. A., Casadaban M. J. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1480–1483. doi: 10.1073/pnas.81.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  14. Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
  15. Kondoh H., Paul B. R., Howe M. M. Use of lambda pMu bacteriophages to isolate lambda specialized transducing bacteriophages carrying genes for bacterial chemotaxis. J Virol. 1980 Sep;35(3):619–628. doi: 10.1128/jvi.35.3.619-628.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. MacNeil D., Howe M. M., Brill W. J. Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes. J Bacteriol. 1980 Mar;141(3):1264–1271. doi: 10.1128/jb.141.3.1264-1271.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mazin A. V., Kuzminov A. V., Dianov G. L., Salganik R. I. Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats. Mol Gen Genet. 1991 Aug;228(1-2):209–214. doi: 10.1007/BF00282467. [DOI] [PubMed] [Google Scholar]
  18. Rayssiguier C., Thaler D. S., Radman M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 1989 Nov 23;342(6248):396–401. doi: 10.1038/342396a0. [DOI] [PubMed] [Google Scholar]
  19. Sternberg N. L., Maurer R. Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium. Methods Enzymol. 1991;204:18–43. doi: 10.1016/0076-6879(91)04004-8. [DOI] [PubMed] [Google Scholar]
  20. Stojiljković I., Trgovcević Z., Salaj-Smic E. Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing. Gene. 1991 Mar 1;99(1):101–104. doi: 10.1016/0378-1119(91)90039-e. [DOI] [PubMed] [Google Scholar]
  21. Taylor R. K., Manoil C., Mekalanos J. J. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J Bacteriol. 1989 Apr;171(4):1870–1878. doi: 10.1128/jb.171.4.1870-1878.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Trun N. J., Silhavy T. J. Characterization and in vivo cloning of prlC, a suppressor of signal sequence mutations in Escherichia coli K12. Genetics. 1987 Aug;116(4):513–521. doi: 10.1093/genetics/116.4.513. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES