The Fordyce Lab

Dropception is a microfluidic platform for creating double emulsion (water-in-oil-in-water) droplets that can be sorted in high-throughput using standard flow cytometers (FACS machines). We recently demonstrated the ability to generate and sort double emulsion droplets without breakage, isolate individual rare droplets of interest in wells of a multiwell plate, and recover all encapsulated nucleic acids, enabling a wide range of novel single-cell multi-omic techniques.

Probing the dark matter of regulatory DNA - Though > 90% of disease-causing variants in the human genome occur outside protein-coding regions, how these non-coding variants impact levels of gene expression remains largely unknown. To address this gap, we are leveraging the MITOMI platform to probe how sequence context outside transcription factor binding sites tunes binding kinetics and thermodynamics.

Dissecting the functional effects of transcription factor mutations - Transcription Factor (TF) proteins bind regulatory DNA sequences to control the expression of downstream genes, and mutations to TFs can affect the affinity and specificity of DNA binding. We are using microfluidic devices to quantitatively measure DNA binding affinities and dissociation kinetics for 1000s of TF-DNA interactions in parallel. This approach provides critical experimental data needed to build computational models of effects of TF mutations required for precision medicine.

HT-MEK: a new kind of enzymology - We have developed a new platform (HT-MEK, for high-throughput microfluidic enzyme kinetics) that enables, for the first time, the rapid and quantitative measurement of multiple parameters for designed enzyme libraries. To date, we have characterized over 1,100 enzyme mutants (measuring >10,000 individual kinetic and thermodynamic parameters), and have identified structurally segregated regions controlling distinct biochemical properties within a model system phosphatase. Ultimately, these measurements will help yield next-generation predictive models of mutational effects required for rational enzyme design and precision medicine.

High-throughput and quantitative measurements of protein stability - Disease-associated protein mutations often act by destabilizing protein structure. In personalized medicine, the majority of disease-associated mutations are of unknown significance, preventing clinical action. We are developing parallel assays to quantify folding stability for thousands of proteins variants in parallel, using next-generation sequencing and fluorescence microscopy. With these approaches, we aim to characterize the protein stability landscape and better understand mechanisms of loss-of-function involved in disease.

BET-seq: ultra-high-throughput measurement of transcription factor-DNA binding affinities - The rapidly decreasing costs of next-generation sequencing (NGS) present an opportunity to sequence and identify millions of DNA molecules in parallel. Here, we paired MITOMI devices with NGS to quantitatively measure transcription factor binding energies for a million sequences in a single experiment. We are currently applying this platform to study how flanking nucleotides outside the canonical binding site can impact TF-DNA binding affinity.

MRBLE-pep - While advances in deep sequencing have dramatically improved our ability to profile how DNA and RNA binding proteins interact with their substrates, we lack similar tools for quantitative, high-throughput investigation of protein-peptide interactions. Here, we synthesize libraries of peptides directly on MRBLEs with a 1:1 linkage between embedded code and peptide sequence, allowing high-throughput measurement of 100 weak protein-peptide interactions in parallel using very little material. In collaboration with the Cyert lab, we are using this platform to understand how human signaling proteins recognize their downstream targets.

BATTLES - T cells can discriminate between self- and non-self-antigens with exquisite sensitivity and specificity: even a single non-self antigen in a sea of 100,000 self antigens. Despite the central importance of this process for immune surveillance, we do not yet understand the mechanisms involved or how to manipulate them to generate new therapies. We are using MRBLEs to allow high-throughput measurements of how T cell activation depends on presented antigen sequence and applied forces.

Systematic characterization of DE droplets – By studying correlations between flow rates, droplet size, and device geometries, we are systematically optimizing DE droplet generation and testing fundamental droplet fluid physics and surfactant compatibility in complex interfaces.

Multi-parameter single-cell analysis – Using sortable double emulsions, we are developing assays to simultaneously screen for rare cell populations and characterize downstream single cell gene expression (RNA-Seq) or epigenomic signatures (ATAC-Seq). The goal of these efforts is to enable new droplet-based techniques that allow tandem ‘omics measurements on the same single cell.

Characterization of individual plant cells at scale – The flexibility of double emulsion droplets permits us to bring single-cell analysis tools to new organisms and new assays. We are developing new ways to measure the transcriptomic profiles and phenotypes of individual rare plant cells of interest.

Organoid models of cancer have revolutionized our ability to recapitulate and observe in vivo cancer processes in a controlled in vitro environment. We are collaborating with Christina Curtis and Calvin Kuo to develop a microwell platform for performing high-throughput phenotyping of organoid heterogeneity across tumor development. This platform allows us to perform time-lapse imaging of thousands of organoids isolated in microwells, followed by computational reconstruction of growth trajectories for each organoid.