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ABSTRACT Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes via extracellular electron transfer (EET). Unfortunately, developing genotype-phenotype relationships for electroactive organisms is challenging because EET is necessarily removed from the cell of origin. Microdroplet emulsions, which encapsulate individual cells in aqueous droplets, have been used to study a variety of extracellular phenotypes but have not been applied to investigate EET. Here, we describe the development of a microdroplet emulsion system to sort and enrich EET-capable organisms from complex populations. We validated our system using the model electrogenShewanella oneidensisand described the tooling of a benchtop microfluidic system for oxygen-limited conditions. We demonstrated the enrichment of strains exhibiting electroactive phenotypes from mixed wild-type and EET-deficient populations. As a proof-of-concept application, we collected samples from iron sedimentation in Town Lake (Austin, TX) and subjected them to microdroplet enrichment. We measured an increase in electroactive organisms in the sorted population that was distinct compared to a population growing in bulk culture with Fe(III) as the sole electron acceptor. Finally, two bacterial species not previously shown to be EET-capable,Cronobacter sakazakiiandVagococcus fessus, were further cultured and characterized for electroactivity. Our results demonstrate the utility of microdroplet emulsions for isolating and identifying EET-capable bacteria.IMPORTANCEThis work outlines a new high-throughput method for identifying electroactive bacteria from mixed populations. Electroactive bacteria play key roles in iron trafficking, soil remediation, and pollutant degradation. Many existing methods for identifying electroactive bacteria are coupled to microbial growth and fitness—as a result, the contributions from weak or poor-growing electrogens are often muted. However, extracellular electron transfer (EET) has historically been difficult to study in high-throughput in a mixed population since extracellular reduction is challenging to trace back to the parent cell and there are no suitable fluorescent readouts for EET. Our method circumvents these challenges by utilizing an aqueous microdroplet emulsion wherein a single cell is statistically isolated in a pico- to nano-liter-sized droplet. Then, via fluorescence obtained from copper reduction, the mixed population can be fluorescently sorted and gated by performance. Utilizing our technique, we characterize two previously unrecognized weak electrogensVagococcus fessusandCronobacter sakazakii. 

Abstract Organic electrochemical transistors (OECTs) are ideal devices for translating biological signals into electrical readouts and have applications in bioelectronics, biosensing, and neuromorphic computing. Despite their potential, developing programmable and modular methods for living systems to interface with OECTs has proven challenging. Here we describe hybrid OECTs containing the model electroactive bacteriumShewanella oneidensisthat enable the transduction of biological computations to electrical responses. Specifically, we fabricated planar p-type OECTs and demonstrated that channel de-doping is driven by extracellular electron transfer (EET) fromS. oneidensis. Leveraging this mechanistic understanding and our ability to control EET flux via transcriptional regulation, we used plasmid-based Boolean logic gates to translate biological computation into current changes within the OECT. Finally, we demonstrated EET-driven changes to OECT synaptic plasticity. This work enables fundamental EET studies and OECT-based biosensing and biocomputing systems with genetically controllable and modular design elements. 

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials.