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Updated: May 24, 2025

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

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Published on: November 25, 2015

Engineering Plasmids with Synthetic Origins of Replication.

Baiyang Liu¹, Xiao Peng¹, Matthew R Bennett^2,3,4

¹Graduate Program in Systems, Synthetic, and Physical Biology, Rice University, Houston, 77005, TX, USA.

Biorxiv : the Preprint Server for Biology

|March 3, 2025

Summary

Researchers engineered synthetic origins of replication (SynORI) for plasmids, enabling customizable copy numbers and modular control. This advance offers a new biotechnology for synthetic biology applications in microorganisms.

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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.