A plasmid system with tunable copy number

Area of Science:

• Synthetic Biology
• Molecular Biology
• Bacteriology

Background:

• Plasmids are essential tools for genetic engineering and recombinant gene expression in bacteria.
• Current plasmid cloning vectors have limited control over copy number, restricting optimization.
• Existing Origins of Replication (ORIs) are derived from naturally occurring plasmids.

Purpose of the Study:

• To introduce novel systems for continuous and fine-tuned control of plasmid copy number.
• To investigate the impact of varying plasmid copy numbers on cellular processes.
• To optimize synthetic biological systems through precise gene expression control.

Main Methods:

• Development of an anhydrotetracycline-controlled plasmid system for tunable copy number.
• Creation of a parallelized assay to generate a wide spectrum of ColE1-based copy number variants.
• Utilizing single-cell timelapse measurements to analyze plasmid dynamics and gene expression variability.

Main Results:

• Achieved continuous control of plasmid copy number from 1 to 800 copies per cell.
• Demonstrated the effects of plasmid copy number on cellular growth, gene expression, and biosynthesis.
• Quantified a linear metabolic burden of 0.063% per plasmid, linking it to DNA synthesis.

Conclusions:

• The developed systems offer precise control over gene expression via plasmid copy number.
• Tuning plasmid copy number is a critical factor for optimizing synthetic biological systems.
• The findings suggest a straightforward relationship between metabolic burden and plasmid DNA synthesis.