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 but are limited by natural replication mechanisms.
• Existing plasmids lack tunability, compatibility, and modularity due to reliance on inherent replication processes.

Purpose of the Study:

• To refactor the natural pMB1 origin of replication for enhanced plasmid control.
• To develop synthetic origins of replication (SynORI) with customizable and independently controlled copy numbers.
• To demonstrate the modularity and signal responsiveness of SynORI for biotechnological applications.

Main Methods:

• Refactoring the pMB1 origin of replication to create tunable components.
• Designing synthetic RNA regulators for independent control of origin activity.
• Engineering SynORI to respond to various environmental signals for multiplexed reporting.
• Constructing and maintaining a library of orthogonal SynORI plasmids in E. coli.

Main Results:

• Customizable plasmid copy numbers were achieved by tuning refactored components.
• Compatible origins with independent copy control using synthetic RNA regulators were created.
• SynORI demonstrated modular engineering for signal-responsive, copy-based reporting.
• A library of six orthogonal SynORI plasmids was successfully co-maintained in E. coli for one week.

Conclusions:

• Synthetic origins of replication (SynORI) offer a feasible and powerful new biotechnology for synthetic biology.
• Refactored and synthetic origins provide enhanced tunability, compatibility, and modularity for plasmid-based systems.
• SynORI enables precise control over plasmid copy number and facilitates complex genetic circuit designs.