Synthetic Integrated In Vitro Transcriptional Regulatory Networks

Abstract

A central goal of systems chemistry is to develop out-of-equilibrium in vitro chemical networks, inspired by cellular genetic regulatory networks (GRNs), that are capable of sensing environmental inputs and directing complex chemical or material dynamics. However, existing out-of-equilibrium in vitro chemistries have limited applicability due to their complex design or restrictive operating conditions. In vitro transcriptional circuits composed of short synthetic DNA complexes, termed genelets, and common, well-characterized enzymes could emulate cellular GRN behavior, however, only small circuits composed of 2-3 genelets have been developed. Furthermore, these previous circuits cannot serve as regulatory networks since they cannot repeatedly change state in response to environmental stimuli. Genelet circuits have also primarily focused on characterizing isolated GRN functional motifs (circuits with specific functions that appear frequently in cellular GRNs) rather than developing larger networks that integrate multiple functional motifs together to enable the sophisticated behavior seen in biology. Here we directly address and overcome the limitations of previous genelet circuits to systematically construct an integrated genelet regulatory network that can change state in response to environmental cues and modularly control the expression of state-specific downstream RNA signals. To build this network, we elucidate design rules for developing responsive and scalable genelet circuits and use predictive kinetic models to guide network design and implementation. This work demonstrates that genelets can be integrated into large networks composed of multiple functional circuit motifs that are capable of dynamic responses to changing environmental inputs and sophisticated regulation of downstream signals. Additionally, RNA, the output of genelet circuits, has been employed within a number of material, chemistry, and biotechnology applications and can be produced at high concentrations, enabling genelet regulatory networks to be readily adapted to a variety of existing systems across a range of scales.