This work draws inspiration from totipotent cellular systems to design smart materials whose compositions and properties can be learned or evolved. Totipotency refers to the inherent genetic potential of a single cell to adapt and produce all types of differentiated cells within an organism. To study this principal and apply it synthetically, tissue-like compartmentalized assemblies are constructed via lipid membrane-separated aqueous droplets in a hydrophobic medium through the droplet interface bilayer (DIB) method. Within our droplets, we explore synthetic totipotency via cell-free reactions including actin polymerization and cell free protein synthesis (CFPS). The transcription and translation of our CFPS reactions are controlled by stimuli-responsive riboswitches (RS). Via this scheme, adaptable material properties and functions are achieved in vitro via protein production from cell-free machinery administered through RS governance. Here, we present thermally or chemically-triggered riboswitches for orthogonal production of representative fluorescent protein products, as well functional proteins. To characterize the material properties of target proteins, we study the formation of polymerized actin shells to stabilize organically-encased droplets and span DIBs. We present a modified protocol for chemically-triggered actin polymerization as well as a thermally triggered actin RS. We characterize theophylline (TP)-triggered production of alpha hemolysin (α-HL) through CFPS and synthesized an organic-soluble trigger that can be sensed from the oil phase by a RS in an aqueous bioreactor droplet. We also demonstrate increased droplet conductivity when CFPS a-HL products are incorporated in DIBs. This interdisciplinary work involves cell culture, gene expression, organic synthesis, vesicle formation, protein quantification, tensiometry, droplet aspiration, microplate fluorescence/absorption experiments, fluorescent microscopy, and electrophysiology. This project is an essential design analysis for creating smart, soft materials using synthetic biology and provides motivation for artificial tissues capable of adapting in response to external stimuli.
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