Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene...

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Bacteriophage-derived systems offer unique tools for microbial gene expression.
• Lactococcus lactis is a key host in food fermentation and biotechnology.
• Efficient heterologous gene expression in L. lactis is crucial for various applications.

Purpose of the Study:

• To develop a novel plasmid-based expression strategy in Lactococcus lactis.
• To exploit lytic bacteriophage features for high-efficiency gene expression.
• To characterize phage-specific expression signals and origins of replication.

Main Methods:

• Cloning of phage phi 31 expression signals using a lacZ reporter system.
• Promoter screening and molecular characterization of phage DNA inserts.
• Construction and induction of recombinant plasmids in L. lactis.
• Assay of beta-galactosidase activity and lacZ mRNA levels.

Main Results:

• Identified and characterized an 888 bp insert from phi 31 containing tandem promoters.
• Demonstrated significant induction of beta-galactosidase and lacZ mRNA upon phi 31 infection.
• Engineered a low-copy-number plasmid with phage origin of replication (ori31) for high-level expression (>2,000 units).
• Showcased synergistic function of phage-inducible promoters and ori31 for rapid, high-efficiency gene expression.

Conclusions:

• A novel phage-inducible expression system was successfully developed in L. lactis.
• The system leverages bacteriophage elements for controlled and efficient heterologous gene expression.
• This strategy holds promise for enhancing biotechnological applications in L. lactis.