Plasmid Vectors for Marker-Free Chromosomal Insertion of Genetic Material in Escherichia coli

  • Sylvie Le Borgne
  • Francisco Bolívar
  • Guillermo Gosset
Part of the Methods in Molecular Biology book series (MIMB, volume 267)


Amethod to achieve the insertion of genetic material into the chromosome of Escherichia coli is described. The method is based on the use of integration vectors from the pBRINTs-rAnbR family. These vectors offer the choice of using the antibiotics chloramphenicol, gentamycin, or kanamycin to select for chromosomal integration events. In addition, it is possible to eliminate these chromosomal antibiotic resistance markers, after integration has taken place. The overall insertion strategy is as follows: a fragment containing the gene(s) to be integrated in the chromosome is inserted into the multiple cloning site of a pBRINTs-rAnbR vector and the resulting plasmid is used to transform E. coli cells. The plasmid is first allowed to replicate in the cell at the permissive temperature of 30°C. Next, the temperature of the culture is raised to 44°C to inhibit plasmid replication and to select for the integrants in the presence of the appropriate antibiotic. Chromosomal excision of the AnbR gene can then be catalyzed by the Cre recombinase that is transiently expressed in the cell from the temperature-sensitive pJW168 plasmid. This plasmid is finally eliminated from the cells by increasing the temperature of the culture to 44°C.

Key Words

Recombinant DNA tools chromosomal integration homologous recombination Cre recombinase loxP unmarked strains 


  1. 1.
    Balbás, P. and Gosset, G. (2001) Chromosomal editing in E. coli: vectors for DNA integration and excision. Mol. Biotechnol. 19, 1–12.PubMedCrossRefGoogle Scholar
  2. 2.
    Balbás, P., Alvarado, X., Bolívar, F., and Valle, F. (1993) Plasmid pBRINT: a vector for chromosomal integration of cloned DNA. Gene 136, 211–213.PubMedCrossRefGoogle Scholar
  3. 3.
    Balbás, P., Alexeyev, M., Shokolenko, I., Bolívar, F., and Valle, F. (1996) A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome. Gene 172, 65–69.PubMedCrossRefGoogle Scholar
  4. 4.
    Le Borgne, S., Palmeros, B., Valle, F., Bolívar, F. and Gosset, G. (1998) pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli. Gene 223, 213–219.PubMedCrossRefGoogle Scholar
  5. 5.
    Palmeros, B., Wild, J., Szybalski, W., Le Borgne, S., Hernández-Chávez, G., Gosset, G., et al. (2000) A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene 247, 255–264.PubMedCrossRefGoogle Scholar
  6. 6.
    Le Borgne, S., Palmeros, B., Bolívar, F. and Gosset, G. (2001) Improvement of the pBRINTTs plasmid family to obtain marker-free chromosomal insertion of cloned DNA in Escherichia coli. BioTechniques 30, 252–256.PubMedGoogle Scholar
  7. 7.
    Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. and Kushner, S. R. (1989) New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol. 171, 4617–4622.PubMedGoogle Scholar
  8. 8.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al. (1995) Short protocols in molecular biology, 3rd ed. John Wiley and Sons Inc., New York, NY.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Sylvie Le Borgne
    • 1
  • Francisco Bolívar
    • 2
  • Guillermo Gosset
    • 2
  1. 1.Departamento de BiotecnologíaInstituto Mexicano del PetóleoMéxico
  2. 2.Instituto de BiotecnologíaUNAMCuernavacaMéxico

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