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Related Experiment Videos

Positive-selection vectors using the F plasmid ccdB killer gene

P Bernard1, P Gabant, E M Bahassi

  • 1Département de Biologie Moléculaire, Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.

Gene
|October 11, 1994
PubMed
Summary

New cloning vectors, pKIL18/19, offer efficient positive selection for recombinant DNA. These plasmids utilize a cytotoxic gene that is only inactivated by foreign DNA insertion, simplifying cloning procedures.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • High-copy-number plasmids like pUC18/19 are widely used for cloning.
  • Positive selection methods simplify the identification of recombinant clones.
  • The ccdB gene product is a potent inhibitor of bacterial gyrase.

Purpose of the Study:

  • To develop novel positive-selection cloning vectors (pKIL18/19) based on pUC18/19.
  • To enhance the efficiency and simplicity of recombinant DNA cloning procedures.
  • To enable high-yield amplification of vectors in specific bacterial hosts.

Main Methods:

  • Construction of pKIL18/19 vectors by fusing the ccdB gene to the lacP promoter and multiple cloning sites.
  • Transformation of Escherichia coli strains (wild-type gyrA+ and gyrA462 mutant) with recombinant and non-recombinant plasmids.

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  • Plating transformed E. coli on rich medium for colony selection.
  • Amplification of vectors in a CcdB-resistant gyrA462 host.

    • Main Results:

    • pKIL18/19 vectors provide highly efficient positive selection for recombinant plasmids.
    • Insertion of foreign DNA into the ccdB gene inactivates its cytotoxic effect, allowing host cell survival.
    • Non-recombinant plasmids containing the active ccdB gene prevent host cell growth.
    • Vectors can be amplified in large quantities using a gyrA462 E. coli strain.

    Conclusions:

    • pKIL18/19 vectors offer a simplified and efficient method for positive selection of recombinant DNA.
    • These vectors are suitable for general cloning and sequencing applications.
    • The use of a CcdB-resistant host facilitates high-yield plasmid preparation.