A Droplet Microfluidics Based Platform for Mining Metagenomic Libraries for Natural Compounds

Abstract

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential.

Click chemistry approaches to expand the repertoire of PEG-based fluorinated surfactants for droplet microfluidics

“…Those observations have practical implications because we have shown that microfluidic droplets can be used to generate highly controlled cell aggregates for high-throughput screening. 67 Remarkably, a simple overlay experiment can reveal some properties of surfactants at molecular scale at the interface.…”

Recent trends in biocatalysis

“…In an interesting approach to screen for proapoptotic compounds, Theodorou et al mixed bacteria in microfluidic droplets with mammalian cells, which could be assayed for apoptosis markers. 468 For the foreseeable future, these complicated screening problems will have to be solved on a case-by-case basis.…”

High-Throughput Optofluidic Acquisition of Microdroplets in Microfluidic Systems

“…The combination of droplet microfluidics and optofluidics, coined hereafter as droplet optofluidics, offers many prospects spanning many academic and industrial fields in biology, chemistry, physics, material, and interface sciences [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. For instance, it enabled the identification of very rare gene sequences [ 12 , 13 , 14 , 15 , 16 , 17 , 18 ], screening of cells or bacteria [ 52 ], membrane proteins inhibitors screening [ 53 ], coupled optical lab-on-chip platform with small angle X-ray scattering (SAXS) [ 54 ], and engineering microparticles for photonics applications [ 55 , 56 ], on-chip multiphasic tunable grating [ 57 ], reconfigurable droplet grating [ 58 ], fluidic Michelson interferometer [ 59 ], droplet grating with polydimethylsiloxane (PDMS) air-lens waveguide setup [ 60 ], 3D and 4D optically fabricated complex geometries [ 61 , 62 ], reconfigurable compound micro-lenses [ 63 ], and countless other possibilities. Previous examples may be considered to be the state-of-the-art, and the domain is flourishing day-by-day to new trends.…”