(540h) Using Photodegradable Hydrogels to Rapidly Screen, Isolate, and Characterize Cells with Rare Phenotypes

The ability to rapidly screen and isolate bacteria with rare function from mutant libraries paves the way for advancements in many applications of microbial biotechnology. However, traditional microbiological screening methods are often time consuming and laborious. Here, we propose a high-throughput screening method to identify and isolate bacterial strains with unique function from mutant library (ML) cell populations for follow up genetic analysis. To this end, a photodegradable poly(ethylene glycol) (PEG) hydrogel was used as a three-dimensional cell scaffold to culture high densities of ML cells within the hydrogel network. ML cells were first encapsulated in a 13µm thick hydrogel layer at a density of 90 cells/mm², allowing for simultaneously monitoring of 28,000 ML cells/ hydrogel. Due to the hydrogel mesh size (10 nm), no transport limitation of nutrients was observed, as the entrapped cells were able to grow into clonal micro-colonies after incubation of the hydrogel in liquid media. For cell extraction, a user controlled, high resolution 365 nm patterned light source was used. This enabled extraction with high levels of spatiotemporal control, allowing one to remove cells in different forms, including free cells, cell aggregates or colonies protected within a PEG coating. Using this approach, we studied the interaction between Agrobacterium tumefaciens C58 and Rhizobium rhizogenes K84, a well-known inhibitor of A. tumefaciens through production of the inhibitory molecule agrocin 84. A. tumefaciens C58 transposon ML cells were encapsulated within the hydrogel, then monitored for rare mutants that show resistance to agrocin 84 by growing in presence of the cell free culture media produced by R. rhizogenes. After identifying rare cells (9/28,000) that developed into microcolonies, cells were extracted for genomic analysis. It was confirmed that resistant colonies had mutations in the acc region of the Ti plasmid. These findings are supported by previous studies that have demonstrated disruption of this region leads to agrocin 84 resistance. This proof of principle study suggests that the proposed screening method, which is simple, affordable, and translatable to microbiology labs, can be used for other applications that require the isolation and study of rare cells with unique phenotype.