(718d) Photoresponsive Hydrogel Membranes for High-Throughput Screening and Isolation of Microbes

Authors: 
Masigol, M., Kansas State University
Hansen, R., Kansas State University
Platt, T., Kansas State University
Fattahi, N., Kansas State University
Barua, N., Kansas State University
Nieves-Otero, P., Kansas State University
van der Vlies, A., Kansas State University
Polymeric materials and interfaces that change properties in response to light exposure have a variety of applications in engineered biomaterials, as they give the user both spatial and temporal control of material properties. In this work, we have engineered photodegradable, polyethylene glycol (PEG) based hydrogels into nanoporous, light-responsive membranes for use in isolation and characterization of microbes [1]. These systems are designed for high-throughput cell screening followed by targeted release and retrieval of individual cells or cell populations that show unique or rare function. The retrieved cells can then be molecularly characterized using ‘Omic’ technologies.

Membranes are formed by combining PEG-o-nitrobenzyl-acrylate macromers with four-armed PEG-tetrathiol crosslinkers, which generate three-dimensional photodegradable hydrogel structures. We have estimated that these hydrogel membranes contain an average mesh size of 14 nm, corresponding to a molecular weight cutoff between 500-670 kDa. This confines bacteria cells but allows for diffusion of biomolecules throughout the gels during culturing and screening steps. Using this material, heterogenous collection of microbial cells can either be encapsulated within the hydrogel itself or confined into microwell compartments, at which point they are examined for function based on growth rate or a fluorescent reporter. Microbial cells that show a rare or unique phenotype are then extracted from the gels using a patterned illumination tool that allows for controlled 365 nm light exposure at high (5 µm) resolution, causing local polymer degradation that triggers the release of cells. Cell populations are then retrieved from the membrane and genotyped.

Using this methodology, we are applying these photo-responsive hydrogel systems for screening microbial communities in microwell formats, for screening large transposon mutant libraries, and for that isolating and characterizing microbes that initiate biofouling processes in wastewater treatment systems. Ultimately, the capability of retrieving phenotypically pure cells from screening interfaces enables one to connect cellular phenotype to genotype, which will aid in the development of microbial communities for engineered applications.

References:

[1] A. J. van der Vlies, N. Barua, P.A. Nieves-Otero, T.G. Platt, and R.R. Hansen, “On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes”, ACS Applied Bio Materials, 2 (1), 266-276 (2019). doi: 10.1021/acsabm.8b00592