Highly Reversible Tunable Thermal-Repressible Split-T7 RNA Polymerases (Thermal-T7RNAPs) for Dynamic Gene Regulation.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Temperature control offers contactless and dynamic regulation of cellular behaviors.
• Existing heat-repressible systems have limitations including complexity, lack of tunability, and delays.

Purpose of the Study:

• To develop a novel, compact, reversible, and tunable thermal-repressible split-T7 RNA polymerase system (Thermal-T7RNAP).
• To enable direct thermal control of T7 RNA polymerase activity within a specific temperature range.

Main Methods:

• Fused temperature-sensitive domains of Tlpa protein with split-T7 RNA polymerase.
• Generated a mutant library with varying thermal performances using an automated screening framework.
• Implemented thermal logic circuitry using the developed mutants.

Main Results:

• Developed Thermal-T7RNAPs enabling direct thermal control of T7 RNA polymerase activity between 30 and 42 °C.
• Extended temperature tunability through a large mutant library and automated screening.
• Demonstrated novel thermal logic circuitry for regulating cell growth and co-culture proportions.

Conclusions:

• The developed Thermal-T7RNAP systems provide a tunable and reversible alternative for thermal regulation in biotechnology.
• This technology expands possibilities for contactless and dynamic control of cellular processes.
• Novel thermal logic circuits were successfully implemented for advanced cellular control.