Integrated Mesoscale Synthetic Regulatory Networks Built from Genelets

Cells are capable of orchestrating complex behaviors, including differentiation and environmental adaptation, using genetic regulatory networks (GRNs) consisting of interconnected genes that regulate one another. Synthetic in vitro chemical reaction networks that mimic cellular GRN dynamics might direct synthetic molecules to exhibit some of the complex dynamic interactions that underlie these complex behaviors. A simple yet powerful toolbox for assembling synthetic GRN analogs are in vitro transcriptional circuits. These circuits comprise short synthetic genelets that are transcribed by viral RNA polymerases and use short nucleic acid sequences to regulate genelet expression, making them straightforward to program and implement. I will describe an updated genelet toolbox that minimizes component crosstalk to enable the construction of large multi-functional regulatory networks. The toolbox comprises >10 unique genelet regulatory motifs that can be interconnected to rapidly create different mesoscale synthetic regulatory networks. Using predictive kinetic models to guide network design and implementation, we assemble genelet networks that (1) exhibit switchable multi-stability, (2) execute temporal programs inspired by cellular developmental pathways, and (3) integrate the behaviors of 1 and 2 to orchestrate state-specific signal expression programs. I will also highlight key challenges that must be addressed to use these networks in downstream applications, including fuel consumption/management and material coupling.