Bacterial Patterning of Soft Materials

Extracellular electron transfer (EET) is an anaerobic respiration mechanism through which electroactive microbes interact with and influence their environment. Using membrane-bound cytochromes, these organisms deposit electrons onto external electron acceptors such as metals and metal oxides. By controlling the expression and activity of EET proteomic elements, it is possible to use electroactive microbes as actuators that confer biological control over abiotic material properties. For example, we have shown that electrons produced by EET-capable organisms can activate exogenous metal catalysts, resulting in radical crosslinking of hydrogel precursors. Additionally, we have also created genetic logic circuits that predictably regulate EET pathway genes and EET flux in <i>Shewanella oneidensis</i> in response to one or more chemical inducers. With the development of these fundamental logic gates, we envision more complex architectures that would allow for spatiotemporal control of hydrogel crosslinking.<br/> <br/>Communities of bacterial cells communicate by secreting chemical signals that act on others in the population in a concentration-dependent manner, usually leading to transcription of specific genes. By engineering strains to utilize these systems, gene expression can be tuned in a spatiotemporally dependent manner. These patterning circuits can spatially control transcription of EET-relevant genes and can be used to pattern crosslinkable materials, such as hydrogels.<br/> <br/>To the end of developing this technology, we have engineered <i>Shewanella oneidensis </i>“sender” cells that, under control of an inducible gene circuit, secrete a diffusible signal. We have also engineered “receiver” cells that express GFP in the presence of the signal. In petri-dish based assays, when fully induced sender cells are placed on top of a lawn of receiver cells and allowed to incubate, a circular “halo” of fluorescence is observed within the signal diffusion gradient produced by the sender cell population. We have found that we can modulate the size and intensity of the fluorescence pattern by varying the induction level of the sender cells. Using image analysis, we were able to quantify the intensity, size, symmetry, and consistency of the pattern.<br/> <br/>The experiment was then repeated with receiver cells that express a key EET pathway protein instead of GFP. A colorimetric reporter of Iron(II) allowed visualization of a “halo” of EET-driven Iron(III) reduction that was very similar in size and shape as the fluorescence experiment, indicating not only spatiotemporal control of EET gene expression, but visual and genetic predictability of EET patterns using the fluorescence system. Future experiments will involve more complex genetic logic within receiver cells to create more intricate patterns as well expanding the reduction substrate scope to dyes and crosslinkable material precursors.<br/> <br/>This technology will lay the groundwork for the engineering of living materials that can react to and interact with complex environments in applications such as chronic wound healing, tissue engineering, device fabrication, and the modeling of developmental signaling systems. Finally, this work will more broadly contribute to the development of EET as a universal interface that allows bacteria to confer genetic control over a large range of material properties.