Protective Proteins Can Improve Cell-Free System Performance in Austere Environments

ABSTRACT: Cell-free gene expression (CFE) technology is an appealing expression chassis for fieldable synthetic biology. Reagents for cell-free protein expression can be preserved, transported, or stored over long periods, even at elevated temperatures. Therefore, cell-free synthetic biology efforts are practical for applications such as fieldable biosensing and decentralized or on-demand therapeutics production in austere environments and at emergency or natural disaster sites. However, these systems still require incubation to operate under standard conditions (e.g., 16 °C to 37 °C), whereas the conditions in the application environment often lie outside these limits. To address this technological gap, we propose adding heat-shock chaperones from diverse organisms to expand the cell-free system’s operating range. We present a method for assessing protective protein candidates, and we demonstrate a 100-fold improvement in fluorescent reporter expression at non-standard temperatures and a widening of the temperature range for system operation by more than 4 °C, as measured by fluorescence from reporter expression. Moreover, we show that dual-chaperone systems can yield higher fluorescence output compared to single-chaperone ones. These chaperone-inspired systems may perform in environments where standard ones fall short, expanding their usability and application potential.