Biology subjects

Endy, D.

Publications and source records attributed to Endy, D..

13 recordsLinked to original sources

bioRxiv2026

Simple and Multiplexed Tracking of Bacterial Growth in Double Emulsion Droplets

Droplet-based microfluidics enable researchers to observe phenotypic heterogeneity within complex biological mixtures through parallel encapsulation of individual samples followed by imaging. Observing or quantifying dynamic heterogeneity remains challenging due to complexities associated with trapping and tracking many individual droplets. Current approaches for time-lapse imaging require specialized devices with droplet traps that limit accessibility and throughput. Here, using readily available materials and software, we demonstrate a simple method for stabilizing and monitoring many, individual droplets for up to 12 hours. We leveraged our method to track bacterial growth within droplets in a high-throughput manner. Our method allows tracking the changes and variation in growth rate within and across droplets, revealing heterogeneity in growth patterns hidden in batch assays. Improving the affordability and throughput of time-dependent phenotyping assays helps to advance biological discovery and biotechnology innovation.

bioengineering↗

bioRxiv2024

Genome-Wide Transcription Response of Staphylococcus epidermidis to Heat Shock and Medically Relevant Glucose Levels

Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. Here, we quantify genome-wide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We find 85 of 2354 genes change up to[~] 34-fold in response to medically relevant changes in glucose concentration (0 mM to 17 mM; adj P value [≤] 0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically relevant change in temperature (37{degrees}C to 45{degrees}C; adj P value [≤] 0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis also revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We further identified 38 glucose-responsive genes as candidate ON or OFF switches for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms.

microbiology↗

bioRxiv2022

Ultra-weak protein-protein interactions can modulate proteome-wide searching and binding

Research on protein-protein interaction (PPIs) tends to focus on high affinity interactions. Weaker interactions (Kd >1M) recently understood as contributing to intracellular phase separation suggest that even-weaker PPIs might also matter in as-yet unknown ways. However, ultra-weak PPIs (Kd >1mM) are not readily accessible by in vivo techniques. Here we use protein electrostatics to estimate PPI strengths and spatially-resolved dynamic simulations to investigate the potential impacts of ultra-weak PPIs within dense protein suspensions. We find that ultra-weak PPIs can drive formation of transient clusters that last long enough to enable enzyme-catalyzed reactions and accelerate the sampling of protein associations. We apply our method to Mycoplasma genitalium, finding that ultra-weak PPIs should be ubiquitous among cytoplasmic proteins. We also predict that the proteome-wide interactome can be shifted to favor binding-dominant ultra-weak PPIs via the introduction of a few charged protein complexes. We speculate that ultra-weak PPIs could contribute to cellular fitness by facilitating sampling and colloidal-scale transport of proteins involved in biological processes, including protein synthesis.

biophysics↗

bioRxiv2021

Molecular transport and packing underlie increasing ribosome productivity in faster growing cells

Faster growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes must increase too, almost doubling by an unknown mechanism. Prior models point to diffusive transport as a limiting factor but surface a paradox: faster growing cells are more crowded, yet crowding slows diffusion. We suspected physical crowding, transport, and stoichiometry, considered together, might reveal a more nuanced explanation. To investigate, we built a first-principles physics-based model of E. coli cytoplasm in which Brownian motion and diffusion arise directly from physical interactions between individual molecules of finite size, density, and physiological abundance. Using our microscopically-detailed model, we predict that physical transport of individual ternary complexes accounts for ~80% of translation elongation latency. We also find that volumetric crowding increases at faster growth even as cytoplasmic mass density remains relatively constant. Despite slowed diffusion, we predict that improved proximity between ternary complexes and ribosomes wins out, illustrating a simple physics-based mechanism for how individual elongating ribosomes become more productive. We speculate how crowding imposes a physical limit on growth rate and undergirds cellular behavior more broadly. Unfitted colloidal-scale modeling offers systems biology a complementary "physics engine" for exploring how cellular-scale behaviors arise from physical transport and reactions among individual molecules.

biophysics↗

bioRxiv2021

Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building

The construction of synthetic cells from lifeless ensembles of molecules is expected to require integration of hundreds of genetically-encoded functions whose collective capacities enable self-reproduction in simple environments. To date the regenerative capacities of various life-essential functions tend to be evaluated on an ad hoc basis, with only a handful of functions tested at once and only successful results typically reported. Here, we develop a framework for systematically evaluating the capacity of a system to remake itself. Using the cell-free Protein synthesis Using Recombinant Elements (PURE) as a model system we apply our framework to evaluate the capacity of PURE, whose composition is completely known, to remake 36 life-essential functions. We find that only 23 of the components can be well tested and that only 19 of the 23 can be remade by the system itself; translation release factors remade by PURE are not fully functional. From both a qualitative and quantitative perspective PURE alone cannot remake PURE. We represent our findings via a standard visual form we call the Pureiodic Table that serves as a tool for tracking which life-essential functions can work together in remaking one another and what functions remain to be remade. We curate and represent all available data to create an expanded Pureiodic Table in support of collective coordination among ongoing but independent synthetic cell building efforts. The history of science and technology teaches us that how we organize ourselves will impact how we organize our cells, and vice versa.

synthetic biology↗

bioRxiv2020

Minicells as a potential chassis for engineering lineage-agnostic organisms

BackgroundGenomes encode for organisms and thus genome synthesis implies the possibility of organismal synthesis, including the synthesis of organisms without constraint to lineage. Current genome-scale engineering projects are focused on minimization, refactoring, or recoding within the context of existing natural lineages. Minicells arise naturally as anucleate cells that are devoid of heritable genetic material but are capable of gene expression. Thus, minicells may serve as a useful starting point for developing lineage-agnostic organisms encoded by newly-designed synthetic genomes. However, the composition and expression capacity of minicells is fixed at the time of their formation. The possibility of reestablishing cellular growth and division starting from minicells and entirely heterologous synthetic genomes is unknown. ResultsWe observed expression and segregation of functional proteins among mixed populations of reproducing cells and so-derived anucleate minicells via fluorescence microscopy. By adapting and integrating established methods of preparation and purification we were able to isolate minicells from a growing population of progenitor cells with a purity of at least 500 minicells per progenitor. We then used heterologous expression of plasmid-encoded green fluorescent protein to estimate the absolute expression capacity of minicells. We found that minicells can support the formation of 4.9 {+/-} 4.6 x 108 peptide bonds prior to exhausting their initial intrinsic expression capacity. ConclusionsMinicells can be produced in large numbers with high purity and can also harbor and express engineered plasmids. The observed variation in minicell gene expression capacity suggests that about 13% of gene-expressing minicells can support the formation of more than one-billion peptide bonds, an amount sufficient to replicate known prokaryotic proteomes. Stated differently, while most minicells would require a sophisticated genetic boot program to first increase minicell-specific expression capacity sufficient to instantiate newly reproducing lineages, a subset of minicells may be able to directly support whole genome, lineage-agnostic organism engineering.

synthetic biology↗

bioRxiv2020

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

bioengineering↗

bioRxiv2019

Enabling community-based metrology for wood-degrading fungi

Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi can enable more direct means of manufacture. However, such practices are often ad hoc and not readily reproducible. We sought to develop standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products. Specifically, we show that a widely-available and globally-regularized consumer product (Pringles) can support the growth of wood-degrading fungi, and that growth on Pringles can be correlated with growth on a fully-traceable and compositionally characterized substrate (NIST Reference Material 8492 Eastern Cottonwood Biomass). So established, five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay. Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial.

synthetic biology↗

bioRxiv2019

Combinatorial analysis of Saccharomyces cerevisiae regulatory elements

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin components has led to the definition and delineation of various regulatory elements. Enhancers, promoters, 5 untranslated regions (5UTR) and transcription terminators/3 untranslated regions (3UTR) have all been defined. However, the specific contributions of each of these features as part of a regulatory unit and the functional communications between these regulatory elements remains under explored.\n\nWe built a combinatorial library of 26 different enhancers, core promoters, 5UTRs and transcription terminators/3UTRs. This library was analyzed with respect to gene expression in order to better understand the interactions between different regulatory elements. In the process we developed new methods to estimate the contribution of individual regulatory parts from just a few simple measurements. Our data show that different pairs of regulatory parts follow specific interaction rules affecting overall activity either positively or negatively. We find that while enhancers are the initiators of gene activity, core promoters modulate the levels of enhancer mediated expression. Cluster analysis based on expression show that TATA-box containing core promoters appear to increase enhancer-driven transcription to a greater extent than TATA-less promoters. Principal component analysis highlight outliers and suggest differences in mechanisms of regulation. These results provide a system to characterize regulatory elements and use these elements in the design of synthetic regulatory circuits.

synthetic biology↗

bioRxiv2019

Synthetic Genetic Codes Designed to Hinder Evolution

One challenge in engineering organisms is guaranteeing system behavior over many generations. Spontaneous mutations that arise before or during use can impact heterologous genetic functions, disrupt system integration, or change organism phenotype. Here, we propose restructuring the genetic code itself such that all point mutations in protein-coding sequences are selected against. Synthetic genetic systems so-encoded should \"fail safely\" in response to many individual spontaneous mutations. We designed a family of such fail-safe codes and analyzed their expected effect on the evolution of engineered organisms via simulation. We predict that fail-safe codes supporting expression of 20 or 15 amino acids could slow the evolution of proteins in so-encoded organisms to 30% or 0% the rate of standard-code organisms, respectively. We also designed quadruplet-codon codes that should be capable of encoding at least 20 amino acids while ensuring all single point mutations in protein-coding sequences are selected against. We show by in vitro experiments that a reduced set of 21 tRNA is capable of expressing a protein whose coding sequence is recoded to use a fail-safe code, whereas a standard-code encoding is not expressed. Our work suggests that a rationally depleted but otherwise natural translation system should yield biological systems with intrinsically reduced evolutionary capacity, and that so-encoded hypoevolvable organisms might be less likely to invade new niches or outcompete native populations.

synthetic biology↗

bioRxiv2019

Abstracted functions for engineering the autonomous growth and formation of patterns

Natural biological patterns arise via the growth, differentiation, death, differential adhesion, communication, and movement of or among cells. Synthetic biologists typically impose explicit genetic control of cell-cell communication and programmable cell state to realize engineered biological patterns. Such engineering approaches do not usually consider the underlying physical properties of individual cells that inevitably contribute to pattern development. To better integrate synthetic genetic systems engineering with natural growth and patterning we derived abstract functions that relate how changes in basic cell properties such as growth rate, length, and radius of curvature result in differences in the curvature, end-point reliability, and texture of borders that define boundaries among growing cell lineages. Each abstracted border function is derived holistically as an emergent consequence of underlying cell physical properties. We experimentally demonstrate control of border curvature to angles of 60{degrees} from initial trajectories, control of end-point variability to within 15{degrees} of desired target endpoints, and control of border texture between 10 to 60 unit cell lengths. In combination with synthetic genetic control systems, we grow arbitrary two-dimensional patterns including phases of the moon, PacMen, and a yinyang-like pattern. Differences between the idealized and observed behavior of abstracted border functions highlight opportunities for realizing more precise control of growth and form, including better integration of synthetic genetic systems with native cellular properties and processes.

synthetic biology↗

bioRxiv2018

Definitive demonstration by synthesis of genome annotation completeness

Bacteriophage oX174 was the first DNA genome to be sequenced. The genome is well studied by classical methods and is known to encode 11 essential genes. At least 23 closely-related Bullavirinae genome sequences are now available. We identified 315 potential open reading frames (ORFs) within the genome via bioinformatic analysis, and a subset of 82 highly-conserved ORFs that have no known gene products or functions. Using genome scale design and synthesis we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved-but-cryptic ORFs. The resulting genome is not viable, as expected. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic-ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ~50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation modulating translation of gene H, a known essential gene, that fully suppressed the fitness defect. Taken together, we conclude that the annotation of ORFs for the oX174 genome is formally complete. Sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

synthetic biology↗

bioRxiv2017

Hands-free control of heterologous gene expression in batch cultures

BackgroundAutonomous cell-based control of heterologous gene expression can simplify batch-culture bioprocessing by eliminating external monitoring and extrinsic control of culture conditions. Existing approaches use auto-induction media, synthetic cell-cell communication systems, or application-specific biosensors. A simpler, resource-efficient, and general-purpose expression control system responsive to common changes during batch culture would be useful.\n\nResultsWe used native E.coli promoters and recombinase-based switches to repurpose endogenous transcription signals for control of heterologous gene expression. Specifically, natural changes in transcription from endogenous promoters result in recombinase expression at different phases of batch culture. So-expressed recombinases invert a constitutive promoter regulating expression of arbitrary heterologous genes. We realized reversible and single-use switching, reduced static and dynamic cell-to-cell variation, and overall expression amplification. We used \"off-the-shelf\" genetic parts and abstraction-based composition frameworks to realize reliable forward engineering of our synthetic genetic systems.\n\nConclusionWe engineered autonomous control systems for regulating heterologous gene expression. Our system uses generic endogenous promoters to sense and control heterologous expression during growth-phase transitions. Our system does not require specialized auto-induction media, production or activation of quorum sensing, or the development of application-specific biosensors. Cells programmed to control themselves could simplify existing bioprocess operations and enable the development of more powerful synthetic genetic systems.

synthetic biology↗