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[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
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.
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.
- 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.