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 thermalrepressible 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.

DNA assembly is a vital process in biotechnology and synthetic biology research, during which DNA plasmids are designed and constructed using bioparts to engineer microorganisms for a wide range of applications. Here, we present an enzymatic homology-based DNA assembly method, SENAX (Stellar ExoNuclease Assembly miX), that can efficiently assemble multiple DNA fragments at ambient temperature from 30 to 37 °C and requires homology overlap as short as 12–18 base pairs. SENAX relies only on a 3′–5′ exonuclease, XthA (ExoIII), followed by Escherichia coli transformation, enabling easy scaling up and optimization. Importantly, SENAX can efficiently assemble short fragments down to 70 bp into a vector, overcoming a key shortcoming of existing commonly used homology-based technologies. To the best of our knowledge, this has not been reported elsewhere using homology-based methods. This advantage leads us to develop a framework to perform DNA assembly in a more modular manner using reusable promoter-RBS short fragments, simplifying the construction process and reducing the cost of DNA synthesis. This approach enables commonly used short bioparts (e.g., promoter, RBS, insulator, terminator) to be reused by the direct assembly of these parts into intermediate constructs. SENAX represents a novel accurate, highly efficient, and automation-friendly DNA assembly method.

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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 and rely on thermal sensitive mRNA or transcription factors which function at low temperatures, lack tunability, suffer delays or 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 fuses temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30-42 °C. We generated a large mutant library with varying thermal performances via 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 expands avenues for thermal control in biotechnology applications.