SUPER: Upcycling Genetic Parts for Precise Gene Expression Control, Leakage Minimization, and Genetic Circuit...

Area of Science:

• Synthetic Biology
• Genetic Engineering
• Molecular Biology

Background:

• Constructing synthetic gene circuits is challenging due to sequence context dependencies, leading to limited dynamic ranges and leaky expression.
• Iterative design-build-test cycles for genetic parts are time-consuming and inefficient.
• Existing genetic parts often lack the desired performance and tunability for complex synthetic biology applications.

Purpose of the Study:

• Introduce SUPER (Synthetic Upcycling Platform for Engineering Regulators), a modular platform to improve genetic device performance.
• Provide an alternative to traditional screening methods for synthetic biology circuits.
• Enhance the control and stability of synthetic genetic circuits.

Main Methods:

• SUPER utilizes small RNA as an add-on controller to modulate gene expression without modifying target regulators.
• The platform was tested by enhancing RNA-, chemical-, temperature-, and protein-responsive regulators.
• SUPER was integrated with a Holin-expressing kill switch and an environmental sensor (TlpA36) to demonstrate its capabilities.

Main Results:

• SUPER enhanced regulator performance by up to 1011% and expanded dynamic ranges up to 22,018.9-fold.
• A SUPER-integrated Holin kill switch maintained stable functionality for over 30 days under selective pressure.
• SUPER enabled the creation of a 2-input chemical- and temperature-responsive kill switch.

Conclusions:

• SUPER offers a systematic framework for upcycling genetic devices, enhancing tunability and performance.
• The platform demonstrates straightforward design, minimal cellular burden, and improved genetic circuit stability.
• SUPER provides a valuable tool for advancing genetic circuit construction in biotechnology and synthetic biology.