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Related Experiment Videos

High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening

D J O'Sullivan1, T R Klaenhammer

  • 1Department of Food Science, North Carolina State University, Raleigh 27695-7624.

Gene
|December 31, 1993
PubMed
Summary

New shuttle cloning vectors offer enhanced genetic manipulation capabilities for industrial bacteria like Lactococcus. These stable, versatile vectors facilitate clone screening and broad bacterial host applicability.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Shuttle vectors are crucial for genetic manipulation in diverse bacterial species.
  • Existing vectors may have limitations in stability or screening efficiency.

Purpose of the Study:

  • To construct and characterize novel high- and low-copy-number shuttle cloning vectors.
  • To assess vector stability and functionality in both Escherichia coli and industrially relevant lactic acid bacteria.

Main Methods:

  • Incorporation of the Escherichia coli P15A plasmid origin of replication into pAM beta 1-derived vectors.
  • Evaluation of vector structural stability in Lactococcus and E. coli.
  • Assessment of erythromycin resistance expression and insertional inactivation of cat and tet genes.

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  • Implementation of blue/white screening using lacZ alpha-complementation.

    • Main Results:

    • Constructed stable high- and low-copy-number shuttle vectors (pTRKH and pTRKL series).
    • Vectors demonstrated structural stability in Lactococcus and E. coli.
    • Expressed erythromycin resistance for selection and offered insertional inactivation for screening.
    • Enabled blue/white screening and were successfully introduced into Lactococcus, Enterococcus, Streptococcus, and Lactobacillus.

    Conclusions:

    • The developed shuttle vectors provide a stable and versatile platform for genetic engineering.
    • These vectors expand the possibilities for genetic manipulation in industrially significant bacteria.
    • The vectors facilitate efficient clone screening and selection across multiple bacterial hosts.