Construction of multilayered gene circuits using de-novo-designed synthetic transcriptional regulators in cell-free...

De-novo-designed synthetic transcriptional regulators have great potential as the genetic parts for constructing complex multilayered gene circuits. The design flexibility afforded by advanced nucleic acid sequence design tools vastly expands the repertoire of regulatory elements for circuit design. In principle, the design space of synthetic regulators should allow for the construction of regulatory circuits of arbitrary complexity; still, the orthogonality and robustness of such components have not been fully elucidated, thereby limiting the depth and width of synthetic circuits.

In this work, we systematically explored the design strategy of synthetic transcriptional regulators, termed switchable transcription terminators. Specifically, by redesigning key sequence domains, we created a high-performance switchable transcription terminator with a maximum fold change of 283.11 upon activation by its cognate input RNA. Further, an automated design algorithm was developed for these elements to improve orthogonality for a complex multi-layered circuit construction. The resulting orthogonal switchable transcription terminators could be used to construct a three-layer cascade circuit and a two-input three-layer OR gate.

We demonstrated a practical strategy for designing standardized regulatory elements and assembling modular gene circuits, ultimately laying the foundation for the streamlined construction of complex synthetic gene circuits.