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A broad-host-range cloning vector transposable to various replicons.

N J Grinter

    Gene
    |January 1, 1983
    PubMed
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    This study presents a novel system for stable DNA insertion into Gram-negative bacterial chromosomes using two plasmids. This method ensures reliable genetic modification in bacteria like E. coli and Pseudomonas.

    Area of Science:

    • Microbiology
    • Molecular Biology
    • Genetic Engineering

    Background:

    • Stable integration of cloned DNA into bacterial chromosomes is crucial for genetic manipulation.
    • Existing methods may lack efficiency or stability in diverse Gram-negative bacteria.
    • Broad-host-range plasmids offer a versatile platform for genetic transfer.

    Purpose of the Study:

    • To develop and validate a robust system for stable chromosomal DNA insertion in Gram-negative bacteria.
    • To demonstrate the efficacy of a two-plasmid system for transposable DNA integration.
    • To assess the stability of integrated DNA sequences across multiple bacterial species.

    Main Methods:

    • Utilized two broad-host-range plasmids: a carrier plasmid with a transposable DNA sequence and a helper plasmid providing transposition functions.

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  • Transferred plasmids between Gram-negative bacteria via conjugation.
  • Enriched for transposition products by exploiting carrier plasmid instability.
  • Stabilized chromosomal insertions by eliminating the helper plasmid.

    • Main Results:

    • Successfully achieved stable insertion of cloned DNA sequences into the chromosomes of Escherichia coli, Methylophilus methylotrophus, and Pseudomonas aeruginosa.
    • Demonstrated high stability of insertions, with less than 0.02% loss per generation.
    • Validated the conjugation and transposition-based system for genetic engineering.

    Conclusions:

    • The described two-plasmid system provides a stable and efficient method for integrating foreign DNA into Gram-negative bacterial chromosomes.
    • This system offers a reliable tool for genetic engineering applications in various Gram-negative species.
    • The high stability of insertions facilitates long-term genetic modifications in bacteria.