Gaining a better understanding of the biological behavior of pathogenic microorganisms is important both in the human and plant sciences. In most systems, microbes exist in communities, consisting of a complex milieu of unique species that may promote or inhibit the pathogen. Because of this inherent complexity, it is difficult to study microbe-microbe interactions in a high-throughput manner with standard microbiology tools. To address the problem, we are developing a microwell array as a high throughput screening tool to study these interactions. The platform is designed to combine a fluorescently-labeled pathogen, i.e. the focal species, with a small number of community members through a stochastic seeding process. Wells are monitored in parallel to identify those wells where focal species growth is inhibited or promoted. However, in order to identify the bacteria responsible for the interaction, it is necessary to retrieve the bacteria from individual microwells of interest for 16S rRNA sequencing. Here, we present the concept and initial experiments of a microwell array with on-demand release of bacteria from selected microwells. As a model bacterium, we use Agrobacterium tumefaciens, the agent of crown gall disease in plants. The key feature of the platform is the attachment of a photo-degradable membrane onto the microwell array that allows for nutrient exchange but prevents motile bacteria from escaping the wells. The photoreactivity of the membrane allows for spatiotemporal control of degradation using a patterned illumination tool, enabling removal above targeted microwells at micron-scale resolution. For fabrication, cells are first seeded into wells using a novel seeding procedure, then the membrane precursor solution containing a photodegradable PEG diacrylate polymer and a four-arm poly(ethylene glycol) (PEG) thiol crosslinker is pipetted over the substrate. A Michael-type thiol-acrylate addition reaction generates the cross-linked polymer membrane. Irradiation of the membrane with light using the Polygon illumination tool degrades the polymer network and opens the microwells allowing bacteria to move out. By simply washing the substrate, the bacteria can be isolated for further characterization. Currently we are developing methods that allow for transfer of bacteria from several wells to agar for further characterization.

On demand release of bacteria from microwell arrays

256th National Meeting (2018)

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