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Gene Replacement in Bordetella pertussis by Transformation with Linear DNA

Bio/Technology volume 8, pages 1025–1029 (1990)Cite this article

Abstract

We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.

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Author notes

  1. G. R. Zealey: Corresponding author.

Authors and Affiliations

  1. Connaught Centre for Biotechnology Research, 1755 Steeles Avenue West, Willowdale, Ontario, Canada, M2R 3T4

    G. R. Zealey, S. M. Loosmore, R. K. Yacoob, S. A. Cockle, L. J. Boux, L. D. Miller & M. H. Klein

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  1. G. R. Zealey
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  2. S. M. Loosmore
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  3. R. K. Yacoob
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  4. S. A. Cockle
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  5. L. J. Boux
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  7. M. H. Klein
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Zealey, G., Loosmore, S., Yacoob, R. et al. Gene Replacement in Bordetella pertussis by Transformation with Linear DNA. Nat Biotechnol 8, 1025–1029 (1990). https://doi.org/10.1038/nbt1190-1025

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  • DOI: https://doi.org/10.1038/nbt1190-1025

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