Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities.

The intertwined adoption of synthetic biology and 3D bioprinting has the potential to improve different application fields by fabricating engineered living materials (ELMs) with unnatural genetically-encoded sense & response capabilities. However, efforts are still needed to streamline the fabrication of sensing ELMs compatible with field use and improving their functional complexity. To investigate these two unmet needs, we adopted a workflow to reproducibly construct bacterial ELMs with synthetic biosensing circuits that provide red pigmentation as visible readout in response to different proof-of-concept chemical inducers. We first fabricated single-input/single-output ELMs and we demonstrated their robust performance in terms of longevity (cell viability and evolutionary stability >15 days, and long-term storage >1 month), sensing in harsh, non-sterile or nutrient-free conditions compatible with field use (soil, water, and clinical samples, including real samples from infected patients). Then, we fabricated ELMs including multiple spatially-separated biosensor strains to engineer: level-bar materials detecting molecule concentration ranges, multi-input/multi-output devices with multiplexed sensing and information processing capabilities, and materials with cell-cell communication enabling on-demand pattern formation. Overall, we showed successful field use and multiplexed functioning of reproducibly fabricated ELMs, paving the way to a future automation of the prototyping process and boosting applications of such devices as in-situ monitoring tools or easy-to-use sensing kits.