Hosted by: Oleg Gang

Cells are capable of orchestrating complex behaviors such as differentiation and stress response using genetic regulatory networks (GRNs) comprising interconnected genes that regulate one another. Many of the key dynamics identified in cellular GRNs have been recapitulated using synthetic chemistries in vitro with an emerging application of autonomous control and dynamic regulation of downstream materials. In this context, in vitro GRN analogs could imbue synthetic materials with the sophisticated behaviors of living systems. In vitro transcriptional circuits, composed of short synthetic genelets, have emerged as a simple yet powerful tool for assembling synthetic GRNs. These circuits utilize only a few inexpensive enzymes and nucleic acids to regulate genelet expression, making them straightforward to program and implement. Yet only small genelet modules that exhibit a single function have been developed. Given cellular GRNs build complexity by integrating many functional modules together, the ability to integrate multiple genelet modules together into larger networks could make it possible to build sophisticated multifunctional GRN analogs. Here we report an updated genelet toolbox that enables the construction of large multifunctional regulatory networks. We develop the toolbox by identifying sources of undesired interactions between network components and designing strategies to mitigate these effects which enables the creation >10 orthogonal genelet node sequences. We assemble these nodes into integrated genelet networks that exhibit either (1) switchable multi-stability inspired by cellular decision making, (2) temporal programs inspired by cellular differentiation pathways, or (3) orchestrate state-specific temporal expression programs. These demonstrations introduce a new class of mesoscale synthetic networks that can orchestrate increasingly complex regulatory processes by design. The genelet regulatory netw