A pipeline for high-throughput microbial isolation, sorting, screening and synthetic community assembly

Microorganisms live as complex communities of different species (e.g. of bacteria and fungi) and play key roles in health and disease in plants and animals, support global nutrient cycles, and represent a source of diverse metabolic pathways and enzymes that can be used to address grand societal challenges in medicine, agriculture and industry. Due to the complexity of microbial communities that can contain millions of cells and thousands of individual species of microbe, separating out individual cells to grow and study them as pure cultures in the laboratory, sequence their genome, understand their physiology, or to screen them for new enzymes and discover new microbial compounds (e.g. antibiotics) is a huge challenge. However, this is now made possible by emerging advanced technologies that separate single cells into microdroplets and select or sort droplets for further laboratory analysis. Here, we will combine three pieces of equipment to develop an experimental pipeline that will enable us to separate single microbial cells from a complex community (or population of cells) and encapsulate them in water/oil or gel droplets where millions of droplets can then be sorted and selected for further screening. This approach uses new advanced research technology that is currently not widely available and will significantly increase the speed at which millions of cells/droplets can be analysed. The equipment will be accessible to a broad user community and will support rapid and significant research advances that will drive a step-change in our ability to isolate microbes into pure culture, to study their biology, and discover new enzymes and microbial compounds that can address grand challenges relating to plant and human health, microbial function, enzyme discovery, screening for antimicrobial resistance, discovery of new antimicrobial compounds, waste management and sustainable energy generation.

Encapsulating individual microbial cells in micrometre-sized droplets prior to high-throughput sorting and selection has huge potential for isolating and culturing recalcitrant microbiota, enzyme discovery, single cell omics, directed evolution, designing and testing synthetic communities and screening for growth rates, enzyme kinetics, mutants and other phenotypes (e.g. antimicrobial resistance). However, current technologies are laborious, time consuming and limited in scale. In this proposal, we will establish a complete pipeline for encapsulation of millions of single microbial cells in liquid or gel-based droplets for high throughput cultivation, sorting and screening using the On-Chip Biotechnologies Droplet Generator, On-chip Droplet Selector and On-chip Sort, enabling selection of single droplets into 96 or 384 well plates for microbial isolation, screening, or single cell analysis and microfluidic sorting of incubated droplets for desired properties (e.g. substrate utilisation). Advantages of the pipeline include microfluidic sorting that enables ultra-low volume reagent quantities, a small laboratory footprint with the capacity to move components of the pipeline into a class 2 biological safety cabinet or an anaerobic chamber as desired, thus expanding the range of potential applications. This workflow will allow early adoption of this emerging advanced research technology, and will serve a broad BBSRC relevant user community focussed on addressing BBSRC-relevant research on plant and human health, microbial function, enzyme discovery, functional metagenomics, screening for antimicrobial resistance, discovery of novel antimicrobial compounds, isolating and characterising environmental microbiota, single cell 'omics', and assembly and characterisation of microbial co-cultures and synthetic microbial communities.