Multilevel Gene Regulation Using Switchable Transcription Terminator and Toehold Switch in Escherichia coli

Nucleic acid-based regulatory components provide a promising toolbox for constructing synthetic biological circuits due to their design flexibility and seamless integration towards complex systems. In particular, small-transcriptional activating RNA (STAR) and toehold switch as regulators of transcription and translation steps have shown a large library size and a wide dynamic range, meeting the criteria to scale up genetic circuit construction. Still, there are limited attempts to integrate the heterogeneous regulatory components for multilevel regulatory circuits in living cells. In this work, inspired by the design principle of STAR, we designed several switchable transcription terminators starting from natural and synthetic terminators. These switchable terminators could be designed to respond to specific RNA triggers with minimal sequence constraints. When combined with toehold switches, the switchable terminators allow simultaneous control of transcription and translation processes to minimize leakage in Escherichia coli. Further, we demonstrated a set of logic gates implementing 2-input AND circuits and multiplexing capabilities to control two different output proteins. This study shows the potential of novel switchable terminator designs that can be computationally designed and seamlessly integrated with other regulatory components, promising to help scale up the complexity of synthetic gene circuits in living cells.