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Abstract Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity.E. coli’s stress response regulon,oxyRS, is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology. 

Abstract BackgroundMicrobial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. ResultsWe created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with thesoxRSoxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulonsoxRS,was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. ConclusionsOur results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems. 

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS , a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices. 

We report a novel strategy for bridging information transfer between electronics and biological systems within microdevices. This strategy relies on our “electrobiofabrication” toolbox that uses electrode-induced signals to assemble biopolymer films at spatially defined sites and then electrochemically “activates” the films for signal processing capabilities. Compared to conventional electrode surface modification approaches, our signal-guided assembly and activation strategy provides on-demand electrode functionalization, and greatly simplifies microfluidic sensor design and fabrication. Specifically, a chitosan film is selectively localized in a microdevice and is covalently modified with phenolic species. The redox active properties of the phenolic species enable the film to transduce molecular to electronic signals ( i.e. , “molectronic”). The resulting “molectronic” sensors are shown to facilitate the electrochemical analysis in real time of biomolecules, including small molecules and enzymes, to cell-based measurements such as cytotoxicity. We believe this strategy provides an alternative, simple, and promising avenue for connecting electronics to biological systems within microfluidic platforms, and eventually will enrich our abilities to study biology in a variety of contexts.