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

Temperature is a physical cue that
is easy to apply, allowing cellular
behaviors to be controlled in a contactless and dynamic manner via
heat-inducible/repressible systems. However, existing heat-repressible
systems are limited in number, rely on thermal sensitive mRNA or transcription
factors that function at low temperatures, lack tunability, suffer
delays, and are overly complex. To provide an alternative mode of
thermal regulation, we developed a library of compact, reversible,
and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs),
which fused temperature-sensitive domains of Tlpa protein with split-T7RNAP
to enable direct thermal control of the T7RNAP activity between 30
and 42 °C. We generated a large mutant library with varying thermal
performances via an automated screening framework to extend temperature
tunability. Lastly, using the mutants, novel thermal logic circuitry
was implemented to regulate cell growth and achieve active thermal
control of the cell proportions within co-cultures. Overall, this
technology expanded avenues for thermal control in biotechnology applications.