Controlling spatial structure in minimal microbial communities by sequential capillary assembly

Bacteria in surface-attached communities often engage in social interactions with neighbouring microbes. Spatial structure within these communities is thought to strongly influence these interactions, yet there is a significant lack of experimental platforms which allow for the tight spatial control of microbial interactions at the microscale, severely limiting our ability to investigate the relationship between spatial structure and community development. Here, we demonstrate a workflow for patterning and growing two bacterial species on a template with high throughput (∼10⁵ patterned cells per template) and micron-scale precision. We demonstrate a methodology for directional sequential capillary assembly of colloidal particles in combination with nanobody-functionalised particles that enable highly specific, bio-orthogonal binding reactions between bacteria and surface deposited particles. Using Staphylococcus aureus and Escherichia coli as model systems, we demonstrate how these organisms can be patterned in any desired spatial configuration where resulting communal growth can be monitored under the microscope. This technique enables careful investigations into the role of initial spatial structure on microbial interactions at low cell density, which is crucial to understanding and manipulating microbial community development.