Characterizing Microbial Communities Using Droplet Microfluidic Technology

Abstract

Bacteria are ubiquitous organisms that play a role in nearly every facet of life from health to the environment. A majority of bacteria live in synergistic communities but many of these bacteria have yet to be cultured and studied in the laboratory. The key to producing new drugs and treating incurable polymicrobial diseases may lie in these uncultured organisms. The objective of this dissertation is to develop a microdroplet platform for co-cultivation and characterization of mixed populations that will enable study of novel bacteria and interactions in microbial community. Previously, our lab successfully developed a proof of concept droplet generation device for co-culturing E. coli. In this dissertation, we continue this work by developing microfluidic technologies to analyze droplets after cultivation. We first developed a device that can separate droplet content. Using streptavidin-functionalized beads, we bound targeted biotinylated microparticles in droplets. Then, using a pneumatically operated separation device, the series of posts in the device trap the beads while the remaining droplet content is removed. The bound targets can be re-encapsulated in a new droplet generated on-chip. We were able to achieve up to 98% purity and 100% yield using this device. Next, we developed a device for separation and dispensing of single droplets. This device is a pneumatically operated two-layer device with a partially closed valve. We tested this device using E. coli cultures in droplets and subsequently amplified the DNA in each isolated droplet. We verified the fidelity of amplification by RT-PCR and whole genome sequencing and showed that the whole genome from each droplet dispensed was successfully amplified. Finally, we demonstrated multi-species amplification in droplets using E. coli and P. putida and verified with RT-PCR both genomes could be amplified regardless of composition. Using this technology, we developed a platform for co-cultivation of human gut bacteria in droplets and used sequencing to elucidate interactions between cultured species. Droplets containing different initial cell numbers and media were incubated under anaerobic conditions. The droplets were then isolated individually for DNA amplification. After amplification, we sequenced the DNA and analyzed one meta-genome to determine functional relationships between two species in a droplet. We found complementary amino acid metabolism pathways for valine, leucine, isoleucine and lysine between the two species as well as differing glycan metabolic pathways in the two species. This platform was re-adapted for cultivation of endosymbiotic bacteria in tunicate. We developed a methodology for extracting endosymbionts from tunicate cells and verified by T-RFLP analysis. Subsequently, the extracted bacteria were cultured in droplets using media from the host extract. This dissertation demonstrates a microfluidic platform for stochastically decomposing complex microbial communities into manageable subsets for studying bacterial interactions using co-cultivation and DNA sequence analysis. This is a powerful tool for high-throughput cultivation and analysis with small sample sizes. Using this platform, we can isolate, culture, and characterize specific interactions between bacteria within a microbial community providing insight that culture-independent metagenomic analysis cannot.