Encapsulating Bacteria in Agarose Microparticles Using Microfluidics for High-Throughput Cell Analysis and Isolation

Abstract

This manuscript describes the high-throughput analysis and isolation of bacterial cells encapsulated in agarose microparticles using fluorescence-activated cell sorting (FACS). Flow-focusing microfluidic systems were used to create monodisperse microparticles that were ~30μm in diameter. The dimensions of these particles made them compatible with flow cytometry and FACS, and the sensitivity of these techniques reduced the incubation time for cell replication before analyses were carried out. The small volume of the microparticles (~1–50 picoliters) minimized the quantity of reagents needed for bacterial studies. This platform made it possible to screen and isolate bacteria, and apply a combination of techniques to rapidly determine the target of biologically active small molecules. As a pilot study, Escherichia coli cells were encapsulated in agarose microparticles, incubated in the presence of varying concentrations of rifampicin, and analyzed using FACS. The minimum inhibitory concentration of rifampicin was determined, and spontaneous mutants that had developed resistance to the antibiotic were isolated via FACS and characterized by DNA sequencing. The β-subunit of RNA polymerase, RpoB, was confirmed as the target of rifampicin, and Q513L was the mutation most frequently observed. Using this approach, the time and quantity of antibiotics required for the isolation of mutants was reduced by 8- and 150-fold, respectively, compared to conventional microbiological techniques using nutrient agar plates. We envision that this technique will have an important impact on research in chemical biology, natural products chemistry, and the discovery and characterization of biologically active secondary metabolites.

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

“…[33] Recently, protocols for high-throughput isolation approaches have been published, but these techniques are still in development. [46][47][48] We were unable to identify reports that described the utilization of highthroughput isolation procedures followed by HTS for BS production; therefore in our opinion, this is a field worth developing. High-throughput isolation techniques also have the potential to assist with the cultivation of currently ''uncultivable'' BS-producing microbes.…”

Microparticles for cell encapsulation and colonic delivery produced by membrane emulsification

“…Eudragit coated microparticles were produced for the colonic release of therapeutics [16,18] or proteins [19], but it has not yet been applied for the coating of encapsulated cells. A variety of materials have been tested for the encapsulation of living cells; alginate [4,6,8,12,15,[20][21][22][23] is the most common and versatile, chitosan [9,23], gelatin [22,24], cellulose [23,25], agarose [23,26], dextran [1], carrageenan [9,12,27], poly(lactide-co-glycolide) (PLGA) [1,23], Poly (Ethylene Glycol) [23,28] all have been used individually, and in blends [1,27]. Chitosan is a proven biocompatible natural polymer produced from natural sources (crustacean shells, fungi, and insects), which has been widely used for cell encapsulation and other pharmaceutical purposes [23,29].…”

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

“…Further, miniaturized methods that use compartmentalization can eliminate competition among species. Cultivation methods that use miniaturization and compartmentalization, including gel microdroplets (14), miniaturized Petri dishes (15), and microfluidics (16)(17)(18)(19), have become increasingly promising as a basis for targeted microbial cultivation and isolation platforms, as they can limit the consumption of precious samples and also control the microenvironment around cells (20). We envisioned implementing targeted cultivation with microfluidics by focusing on two goals.…”