[Construction and identification of exogenous gene expression system in Lactococcus lactis]

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lactococcus lactis is a widely used lactic acid bacterium in food fermentation.
• Developing efficient exogenous gene expression systems is crucial for its genetic manipulation and biotechnological applications.

Purpose of the Study:

• To construct and characterize a novel exogenous gene expression system in Lactococcus lactis.
• To establish functional plasmids for expressing foreign genes in this bacterial host.

Main Methods:

• Utilized PCR-based gene assembly to synthesize key genetic elements including the P59 promoter, USP45 signal peptide, and ribosome binding site.
• Constructed recombinant plasmids pNBC1000 and pNBC2000, incorporating the Streptococcus pyogenes M6 protein gene.
• Verified expression capability by cloning the enhanced green fluorescent protein (EGFP) gene and observing fluorescence using laser scanning confocal microscopy.

Main Results:

• Successful construction of plasmids pNBC1000 and pNBC2000.
• Demonstrated exogenous gene expression in Lactococcus lactis, evidenced by visualized green fluorescence in bacteria containing the pNBC2000-EGFP construct.
• Confirmed no fluorescence in control groups (pNBC2000 and pBS-EGFP), indicating successful expression system functionality.

Conclusions:

• The developed pNBC1000 and pNBC2000 plasmids are functional for exogenous gene expression in Lactococcus lactis.
• The system incorporates essential elements like the P59 promoter and USP45 signal peptide for effective expression.
• This advancement provides a valuable tool for genetic engineering and metabolic pathway manipulation in Lactococcus lactis.