The missing link: Single cell culture of the human microbiota links the bacterial metabolome to bacterial genomes and biosynthetic gene clusters

The Science and Technology behind Design Pharma

Microbial metabolites are important mediators of microbe-microbe and host-microbe interactions and represent a rich resource for drug discovery. In our last article, we described the tools we are using to bioinformatically determine biosynthetic gene clusters of interest that encode such molecules with drug-like properties. Today's article provides an overview of our microfluidic platform for culturing the dark matter of the human microbiota for drug discovery.

Droplet microfluidics is a technique that utilizes 2 immiscible phases: a continuous flowing oil phase and a dispersed aqueous phase. With the help of the oil phase, small aqueous droplets are generated at high speed in micrometer-channel devices. A biocompatible surfactant dissolved in the oil forms an amphiphilic monolayer at the interface of the water and oil, thereby stabilizing the droplets. Hence, droplets are small, stable vessels in which experiments can be conducted. With Design Pharma’s proprietary microbiome-on-a-chip technology platform, droplets can be created on average at speeds of 10,000 droplets/s. Droplet volumes are precisely adjusted in a range down to a few picoliters and up to a few nanoliters, depending on the experimental requirements. Due to the dramatic miniaturization, droplet populations easily comprising 100 million individual droplets are processed within hours, at the same time keeping the consumption of valuable reagents to a minimum.

This enables the encapsulation of single bacterial cells in growth media containing droplets at an amazing rate at which the cells can grow into clonal populations. Due to our high droplet generation throughput and low droplet volume, we generate and incubate droplet populations with 2.5 x 10^7?droplets, which have a total volume of only 500 μl. This means that we encapsulate 1.25 x 107?single cells in droplets, providing an exceptional sampling depth for the microbiota. Due to the extremely small volume, the bacterial samples can be divided among several growth media and also allow full atmospheric control of the droplet culture, e.g. under strictly anaerobic conditions.

Unlike conventional microbiological methods, single cell culture in the microfluidic droplet environment preserves a much larger portion of the original microbiota diversity. Accordingly, single cell culture and deep sampling of the human microbiota allows Design Pharma to phenotypically characterize bacterial species and their culture supernatants directly in bioactivity assays such as G protein-coupled receptor assays. Hits from bioactivity assays can then be linked to biosynthetic gene clusters encoded in the bacterial genome by sequencing the genome and assessing molecules produced by the specific species using MassSpec and other tools. Thus, our unique ability to comprehensively culture the dark matter of the microbiota gives us direct access to the molecules with drug-like potential encoded by the vast diversity of the microbiota.