Engineering plasmids with synthetic origins of replication

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Plasmids are essential for delivering engineered DNA to microorganisms.
• Natural plasmid replication limits tunability, compatibility, and modularity.

Purpose of the Study:

• To refactor natural plasmid origins for customizable copy numbers.
• To develop compatible synthetic origins of replication (SynORI) with independent copy control.
• To demonstrate modularity and signal responsiveness of SynORI for multiplexed reporting.

Main Methods:

• Refactoring the natural pMB1 origin of replication.
• Designing synthetic RNA regulators for copy number control.
• Engineering SynORI for modularity and signal responsiveness.
• Creating and co-maintaining a library of compatible SynORI plasmids in E. coli.

Main Results:

• Created plasmids with tunable, customizable copy numbers.
• Developed compatible origins using synthetic RNA regulators.
• Demonstrated SynORI's modularity for signal-based copy control and reporting.
• Successfully co-maintained a library of 6 compatible SynORI plasmids in E. coli for one week.

Conclusions:

• Established the feasibility of SynORI for creating advanced plasmids.
• SynORI offers a versatile biotechnology platform for synthetic biology.
• This work enhances DNA delivery systems for microbial engineering.