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Related Experiment Videos

[Expression vector pLF22 for the lactic acid bacteria].

B V Tarakanov1, A A Iakovleva, T A Nikolicheva

  • 1All-Russia State Institute of the Physiology, Biochemistry, and Feed of Livestock, Borovsk, Kaluga Oblast, 249013 Russia.

Mikrobiologiia
|June 17, 2004
PubMed
Summary

A new expression vector, pLF22, was developed for lactic acid bacteria and other microbes. This vector enables constitutive gene expression and was validated using beta-galactosidase and somatotropin releasing factor (SRF) genes.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Development of versatile expression vectors is crucial for genetic engineering in bacteria.
  • Lactic acid bacteria (LAB) are important probiotics with potential for therapeutic applications.
  • Efficient gene expression systems are needed for functional studies and applications in diverse bacterial hosts.

Purpose of the Study:

  • To construct and characterize a novel expression vector, pLF22, for use in lactic acid bacteria and other bacterial species.
  • To ensure constitutive transcription of cloned genes within the vector system.
  • To demonstrate the vector's functionality in both Gram-negative and Gram-positive bacteria, including probiotic strains.

Main Methods:

  • Construction of the pLF22 expression vector using a replicon from Lactobacillus fermentum cryptic plasmid pLF1311 and a lacZ' multiple cloning site.

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  • Integration of the multiple cloning site with the plasmid replication (rep) operon for constitutive transcription.
  • Verification of vector efficiency by expressing the beta-galactosidase gene in Escherichia coli and the somatotropin releasing factor (SRF) gene in probiotic lactobacilli and enterococci.
  • Assessment of the physiological and histological effects of a recombinant strain expressing SRF in laboratory animals.

    • Main Results:

    • The pLF22 vector facilitates constitutive transcription of cloned genes lacking transcription terminators.
    • The vector is functional in a broad range of Gram-positive and Gram-negative bacteria, including probiotic strains.
    • Successful expression of beta-galactosidase and SRF genes was achieved in E. coli and probiotic bacteria, respectively.
    • Dietary administration of SRF-expressing recombinant bacteria modulated physiological, anthropometric, and histological parameters in animals.

    Conclusions:

    • The pLF22 vector is a versatile tool for gene expression in diverse bacterial hosts, including important probiotic species.
    • Constitutive transcription provided by pLF22 enables efficient heterologous gene expression.
    • The successful expression of SRF and its biological effects highlight the potential of this vector system for developing novel probiotic applications.