Shuttle vectors developed from Streptococcus thermophilus native plasmid

Area of Science:

• Molecular Biology
• Microbiology
• Genetic Engineering

Background:

• Streptococcus thermophilus is a key bacterium in dairy fermentation.
• Efficient genetic tools are needed for manipulating S. thermophilus strains.
• Native plasmids can serve as a basis for developing novel cloning vectors.

Purpose of the Study:

• To develop novel shuttle vectors based on the pER8 plasmid from S. thermophilus.
• To create vectors capable of replication and selection in both Escherichia coli and S. thermophilus.
• To engineer vectors with selectable markers for genetic manipulation.

Main Methods:

• Construction of pMEU-series shuttle vectors by combining pER8 elements with E. coli and Staphylococcus plasmids.
• Introduction of replication origins and antibiotic resistance genes (bla, erm, cat).
• Transformation efficiency assays in E. coli and S. thermophilus.
• Stability tests in both host systems.

Main Results:

• Developed pMEU5-10 shuttle vectors (5.7-6.9 kb) functional in E. coli and S. thermophilus.
• Achieved high transformation frequencies: ~10^5-10^6 CFU/µg DNA in E. coli and ~10^3 CFU/µg DNA in S. thermophilus.
• Demonstrated high stability of the vectors in both bacterial hosts.
• pMEU9 and pMEU10 offer utility for insertional inactivation-based clonal selection.

Conclusions:

• The pMEU-series vectors are effective tools for molecular cloning in S. thermophilus and E. coli.
• These shuttle vectors enhance genetic manipulation capabilities for S. thermophilus research.
• The developed vectors provide stable and efficient means for bacterial strain engineering.