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Introduction:Molecular communication is the transfer of information encoded by molecular structure and activity. We examine molecular communication within bacterial consortia as cells with diverse biosynthetic capabilities can be assembled for enhanced function. Their coordination, both in terms of engineered genetic circuits within individual cells as well as their population-scale functions, is needed to ensure robust performance. We have suggested that “electrogenetics,” the use of electronics to activate specific genetic circuits, is a means by which electronic devices can mediate molecular communication, ultimately enabling programmable control. Methods:Here, we have developed a graphical network model for dynamically assessing electronic and molecular signal propagation schemes wherein nodes represent individual cells, and their edges represent communication channels by which signaling molecules are transferred. We utilize graph properties such as edge dynamics and graph topology to interrogate the signaling dynamics of specific engineered bacterial consortia. Results:We were able to recapitulate previous experimental systems with our model. In addition, we found that networks with more distinct subpopulations (high network modularity) propagated signals more slowly than randomized networks, while strategic arrangement of subpopulations with respect to the inducer source (an electrode) can increase signal output and outperform otherwise homogeneous networks. Discussion:We developed this model to better understand our previous experimental results, but also to enable future designs wherein subpopulation composition, genetic circuits, and spatial configurations can be varied to tune performance. We suggest that this work may provide insight into the signaling which occurs in synthetically assembled systems as well as native microbial communities. 

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.