Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes

Abstract

Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 μm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 μm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

“…As reported previously 19 , it was observed that cell density of 7.26 × 10 7 CFU/mL in the crosslinking buffer correlates to a density of ~ 90 cells/mm 2 encapsulated across the hydrogel. The prepared hydrogel can be incubated in media for culture.…”

“…The separation process in both formats has been successfully used to isolate bacteria that display unique growth behavior for downstream genotyping after culture and microscopic observation, a critical capability for connecting cell genotype to phenotype. To date, genomic characterizations of bacteria extracted from these interfaces have included 16S amplicon sequencing to identify multi-species collections of bacteria from environmental microbiomes that generate emergent growth behavior 18 , and for whole-genome sequencing to successfully identify genetic mutations that cause rare growth profiles in cells present within mutant libraries 19 .…”

“…After 365 nm UV light exposure, hydrogels were labeled with fluorescein-5-maleimide for fluorescence imaging. Reprinted (adapted)with permission from Fattahi et al19 Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.…”

“…This colony is also removed, resulting in cross-contamination. Reprinted (adapted) with permission from Fattahi et al19 Copyright 2020 American Chemical Society. Please click here to view a larger version of this figure.…”

An integrated microfluidic chip for alginate microsphere generation and 3D cell culture

“…12,[17][18][19] Hydrogel microspheres are widely used for microbial cell protection against the harsh conditions of reaction media such as variable temperature, pH and shear stress. 10,20 Moreover, hydrogel microspheres provide an excellent solution for cell culture and lineage, due to their large surface area, small regular shape, and extensive adjustability. 12,19,21 A single hydrogel microsphere can be used for capturing of single or few cells followed by monitoring of growth and phenotypic characterization.…”