A5085863617- Innovative Microfluidic and Catalytic Techniques Innovation
- RNA and protein synthesis mechanisms
- Genomics and Chromatin Dynamics
- Microfluidic and Capillary Electrophoresis Applications
- Electrowetting and Microfluidic Technologies
- RNA Research and Splicing
- Protein Structure and Dynamics
- Single-cell and spatial transcriptomics
- 3D Printing in Biomedical Research
- Chemical Synthesis and Analysis
- Advanced biosensing and bioanalysis techniques
- Gene Regulatory Network Analysis
- Microfluidic and Bio-sensing Technologies
- Force Microscopy Techniques and Applications
- Biochemical and Molecular Research
- Monoclonal and Polyclonal Antibodies Research
- Advanced Biosensing Techniques and Applications
- Fungal and yeast genetics research
- CRISPR and Genetic Engineering
- Microtubule and mitosis dynamics
- Viral Infectious Diseases and Gene Expression in Insects
- Signaling Pathways in Disease
- Enzyme Structure and Function
- Microbial Metabolic Engineering and Bioproduction
- Biochemical and Structural Characterization
Stanford University
2016-2025
Chan Zuckerberg Initiative (United States)
2017-2025
Bioengineering Center
2017-2023
Palo Alto University
2023
Chan Zuckerberg Biohub San Francisco
2018-2022
Rockford University
2017
Foundry (United Kingdom)
2017
University of California, San Francisco
2010-2015
Howard Hughes Medical Institute
2010-2015
University of Utah
1984
Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency meet current challenges in medicine engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, characterization more than 1500 enzyme variants per experiment. For 1036 mutants the alkaline phosphatase PafA (phosphate-irrepressible Flavobacterium), we performed 670,000 reactions determined 5000...
Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate factor (TF)-DNA affinities apparent on-rates by about 70-fold directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. maximize the number of weakly preferred microstates near target sites, thereby increasing density, well predicted statistical mechanics....
Adoptive cell therapy using engineered T receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured can enhance the efficacy of TCR-T also cross-react with off-target resulting in organ immunopathology. We developed an alternative strategy isolate TCR mutants that exhibited high activation signals coupled low-affinity pMHC binding...
Abstract Droplet microfluidics enables kHz screening of picoliter samples at a fraction the cost other high-throughput approaches. However, generating stable droplets with desired characteristics typically requires labor-intensive empirical optimization device designs and flow conditions that limit adoption to specialist labs. Here, we compile comprehensive droplet dataset use it train machine learning models capable accurately predicting geometries required generate aqueous-in-oil...
The ability of kinesin to travel long distances on its microtubule track without dissociating has led a variety models explain how this remarkable degree processivity is maintained. All these require that the two motor domains remain enzymatically "out phase," behavior would ensure that, at any given time, one strongly attached microtubule. maintenance coordination over many mechanochemical cycles never been explained, because key steps in cycle could not be directly observed. We have...
Abstract Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across massively parallel (5184) nano-well array, yielding nearly 20-fold improvement throughput (up ~1800 cells/chip, 4–5 h on-chip processing time) library preparation cost (~81¢ per cell) compared prior microfluidic implementations. We apply...
The human fungal pathogen Candida albicans can switch between two phenotypic cell types, termed 'white' and 'opaque'. Both types are heritable for many generations, the occurs epigenetically, that is, without a change in primary DNA sequence of genome. Previous work identified six key transcriptional regulators important white-opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, Ahr1. In this work, we describe structure network specifies white opaque governs ability to them. particular, use...
We have developed a novel workflow (sdDE-FACS, s̲ingle d̲roplet D̲ouble E̲mulsion FACS) that allows robust production, screening, and sorting of single double emulsion droplets with complete nucleic acid recovery.
Understanding how proteins discriminate between preferred and non-preferred ligands ('selectivity') is essential for predicting biological function a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study variants promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity selectivity, yielding >1700 Kds >500 rate constants in complex with multiple sequences. Twenty-two 240...
Spectrally encoded fluorescent beads are an attractive platform for assay miniaturization and multiplexing in the biological sciences. Here, we synthesize hydrophilic PEG–acrylate polymer with lanthanide nanophosphors using a fully automated microfluidic synthesis device. These by including varying amounts of two relative to third reference nanophosphor generate 24 distinct ratios. codes differ less than 3% from their target values can be distinguished each other error rate <0.1%. The bead...
Abstract Motivation Transcription factors bind regulatory DNA sequences in a combinatorial manner to modulate gene expression. Deep neural networks (DNNs) can learn the cis-regulatory grammars encoded associated with transcription factor binding and chromatin accessibility. Several feature attribution methods have been developed for estimating predictive importance of individual features (nucleotides or motifs) any input sequence its output prediction from DNN model. However, these do not...
Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements TF-DNA binding energies can improve the accuracy predictions TF occupancy and downstream vivo shed light on how transcriptional networks rewired throughout evolution. Here, we present a sequencing-based assay analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable providing quantitative...
Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects biology. In this work, we identify a transcriptional regulator from human fungal pathogen Candida albicans binds DNA specifically but has no detectable homology with any previously described DNA- or RNA-binding protein. This protein, named White–Opaque Regulator 3 (Wor3), regulates white–opaque switching, ability C. to switch...
Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions other proteins drives cellular signaling. Mapping the energy landscapes these interactions is essential for deciphering and perturbing signaling networks but challenging due their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies library peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we...
In the past five years, droplet microfluidic techniques have unlocked new opportunities for high-throughput genome-wide analysis of single cells, transforming our understanding cellular diversity and function. However, field lacks an accessible method to screen sort droplets based on phenotype upstream genetic analysis, particularly large complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow precise single-cell encapsulation into picoliter-scale double...
Abstract Sequence-specific activation by transcription factors is essential for gene regulation 1,2 . Key to this are domains, which often fall within disordered regions of 3,4 and recruit co-activators initiate 5 These interactions difficult characterize via most experimental techniques because they typically weak transient 6,7 Consequently, we know very little about whether these promiscuous or specific, the mechanisms binding, how tune strength activation. To address questions, developed...
A quantitative understanding of how transcription factors interact with genomic target sites is crucial for reconstructing transcriptional networks in vivo. Here, we use Hac1, a well-characterized basic leucine zipper (bZIP) factor involved the unfolded protein response (UPR) as model to investigate interactions between bZIP and their sites. During UPR, accumulation proteins leads unconventional splicing subsequent translation HAC1 mRNA, followed by UPR genes. Initial candidate-based...
Multilayer soft lithography (MSL) provides a convenient and low-cost method for fabricating poly(dimethyl siloxane) (PDMS) microfluidic devices with on-chip valves automated precise control of fluid flow. MSL casting molds flow channels typically incorporate small patches rounded positive photoresist at valve locations to achieve the cross-sectional profile required these function properly. Despite importance features device performance, comprehensive characterization how rounding process...
The ability to produce and distinguish microparticles containing >1100 spectral codes via the ratiometric incorporation of lanthanide nanophosphors is demonstrated. It shown that 98% these microparticles, which are termed MRBLEs (microspheres with barcode encoding), can be assigned a code >99.99% confidence, representing largest purely set produced date. As service our authors readers, this journal provides supporting information supplied by authors. Such materials peer reviewed may...