The ability to extract specific portions of microbial populations may be useful for many applications. We aim to develop novel materials and methodologies allowing the isolation of specific cell populations with desired or unique functions. We use a photodegradable, poly (ethylene glycol) hydrogel as a three-dimensional cell culture platform to screen, isolate, and sequence pathogens capable of growing in the presence of an antagonizing organism. A controllable, patterned, high-resolution light source allows for spatial and temporal control of release of targeted cell micro-colonies within the hydrogel for sequencing to determine the genetic pathways responsible for resistance to antagonism. We are currently studying interactions between Agrobacterium tumefaciens C58 and the biocontrol agent Rhizobium rhizogenes K84. R. rhizogenes K84 is a potent antagonist of many strains of pathogenic A. tumefaciens, the causative agent of crown gall disease. Bacteria seeded into hydrogels form clonal micro-colonies within the hydrogel, confirming that the polymer network allows nutrient exchange and cell growth. We also show that targeted micro-colonies can be extracted from the hydrogel using the patterned illumination tool (Fig.1). We were able to extract and culture a rare C58 mutant able of growing in the presence of K84. Our future aim is to use these approaches in a high-throughput screen in which tens of thousands of mutants can be examined for their resistance to K84 antagonism within a single hydrogel. Doing this will allow us to identify and map rare mutations conferring resistance to K84 antagonism.

Screening diffusive, antagonistic bacterial interactions using photoreleasable hydrogels

2019 Spring National Meeting

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