Biology subjects

Mangan, N. M.

Publications and source records attributed to Mangan, N. M..

8 recordsLinked to original sources

bioRxiv2025

GCAD: a Computational Framework for Mammalian Genetic Program Computer-Aided Design

Genetic programs can direct living systems to perform diverse, pre-specified functions. As the library of parts available for building such programs continues to expand, computation-guided design is increasingly helpful and necessary. Predictive models aid the challenging design process, but iterative simulation and experimentation are intractable for complex functions. Computer-aided design accelerates this process, but existing tools do not yet capture the behavior of mammalian-specific parts and population-level effects needed for mammalian synthetic biologists. To address these needs, we developed a framework for mammalian genetic program computer-aided design. Starting with a user-defined design specification to quantify circuit performance, the framework uses a genetic algorithm to search through possible designs. Circuit space is defined by a library of experimentally characterized parts and dynamical systems models for gene expression in a heterogeneous cell population. We developed this genetic algorithm using a directed graph-based formulation with biologically constrained rules to explore regulatory connections and parts. We evaluated the framework for design problems of varying complexity, including programs we describe as an amplifier, signal conditioner, and pulse generator, demonstrating that the algorithm can successfully find optimal circuit designs. Finally, we experimentally evaluated selected circuits, demonstrating the path from a predicted circuit design to experimental testing and highlighting the importance of characterization for enabling predictive design. Overall, this framework establishes general approaches that can be refined and expanded, accelerating the design and implementation of mammalian genetic programs.

synthetic biology↗

bioRxiv2024

Bicoid-nucleosome competition sets a concentration threshold for transcription constrained by genome replication

Transcription factors (TFs) regulate gene expression despite constraints from chromatin structure and the cell cycle. Here we examine the concentration-dependent regulation of hunchback by the Bicoid morphogen through a combination of quantitative imaging, mathematical modeling and epigenomics in Drosophila embryos. By live imaging of MS2 reporters, we find that, following mitosis, the timing of transcriptional activation driven by the hunchback P2 (hb P2) enhancer directly reflects Bicoid concentration. We build a stochastic model that can explain in vivo onset time distributions by accounting for both the competition between Bicoid and nucleosomes at hb P2 and a negative influence of DNA replication on transcriptional elongation. Experimental modulation of nucleosome stability alters onset time distributions and the posterior boundary of hunchback expression. We conclude that TF-nucleosome competition is the molecular mechanism whereby the Bicoid morphogen gradient specifies the posterior boundary of hunchback expression.

developmental biology↗

bioRxiv2024

Developing, characterizing and modelingCRISPR-based point-of-use pathogen diagnostics

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

synthetic biology↗

bioRxiv2022

Mega-scale experimental analysis of protein folding stability in biology and protein design

Advances in DNA sequencing and machine learning are illuminating protein sequences and structures on an enormous scale. However, the energetics driving folding are invisible in these structures and remain largely unknown. The hidden thermodynamics of folding can drive disease, shape protein evolution, and guide protein engineering, and new approaches are needed to reveal these thermodynamics for every sequence and structure. We present cDNA display proteolysis, a new method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of ~850,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 354 natural and 188 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate, and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability. One-Sentence SummaryMassively parallel measurement of protein folding stability by cDNA display proteolysis

biophysics↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

synthetic biology↗

bioRxiv2021

Physical constraints on growth dynamics guide C. elegans developmental trajectories and animal shape

Growth control establishes organism size, requiring mechanisms to sense and adjust growth during development. Studies of single cells revealed that size homeostasis uses distinct control methods. In multicellular organisms, mechanisms that regulate single cell growth must integrate control across organs and tissues during development to generate adult size and shape. We leveraged the roundworm Caenorhabditis elegans as a scalable and tractable model to collect precise growth measurements of thousands of individuals, measure feeding behavior, and quantify changes in animal size and shape during a densely sampled developmental time course. As animals transitioned from one developmental stage to the next, we observed changes in body aspect ratio while body volume remained constant. Then, we modeled a physical mechanism by which constraints on cuticle stretch could cause changes in C. elegans body shape. The model-predicted shape changes are consistent with those observed in the data. Theoretically, cuticle stretch could be sensed by the animal to initiate larval-stage transitions, providing a means for physical constraints to influence developmental timing and growth rate in C. elegans. HighlightsO_LIBody size measurements of thousands of animals in a dense developmental time course C_LIO_LIGrowth rate exhibits nonlinear dynamics in both length and width C_LIO_LIChanges in body shape but not volume occur during periods of increased quiescence C_LIO_LIDynamics of animal shape consistent with a length-based threshold in cuticle stretch C_LIO_LIModeling of cuticle stretch dynamics suggests a novel mode for growth control C_LI

developmental biology↗

bioRxiv2021

Diffusion barriers and adaptive carbon uptake strategies enhance the modeled performance of the algal CO2-concentrating mechanism

Many photosynthetic organisms enhance the performance of their CO2-fixing enzyme Rubisco by operating a CO2-concentrating mechanism (CCM). Most CCMs in eukaryotic algae supply concentrated CO2 to Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer an algal CCM into crops that lack a CCM to increase yields. To advance our basic understanding of the algal CCM, we develop a chloroplast-scale reaction-diffusion model to analyze the efficacy and the energy efficiency of the CCM in the green alga Chlamydomonas reinhardtii. We show that achieving an effective and energetically efficient CCM requires a physical barrier such as thylakoid stacks or a starch sheath to reduce CO2 leakage out of the pyrenoid matrix. Our model provides insights into the relative performance of two distinct inorganic carbon uptake strategies: at air-level CO2, a CCM can operate effectively by taking up passively diffusing external CO2 and catalyzing its conversion to HCO3-, which is then trapped in the chloroplast; however, at lower external CO2 levels, effective CO2 concentration requires active import of HCO3-. We also find that proper localization of carbonic anhydrases can reduce futile carbon cycling between CO2 and HCO3-, thus enhancing CCM performance. We propose a four-step engineering path that increases predicted CO2 saturation of Rubisco up to seven-fold at a theoretical cost of only 1.5 ATP per CO2 fixed. Our system-level analysis establishes biophysical principles underlying the CCM that are broadly applicable to other algae and provides a framework to guide efforts to engineer an algal CCM into land plants. Significance StatementEukaryotic algae mediate approximately one-third of CO2 fixation in the global carbon cycle. Many algae enhance their CO2-fixing ability by operating a CO2-concentrating mechanism (CCM). Our model of the algal CCM lays a solid biophysical groundwork for understanding its operation. The models consistency with experimental observations supports existing hypotheses about the operating principles of the algal CCM and the functions of its component proteins. We provide a quantitative estimate of the CCMs energy efficiency and compare the performance of two distinct CO2 assimilation strategies under varied conditions. The model offers a quantitative framework to guide the engineering of an algal CCM into land plants and supports the feasibility of this endeavor.

biophysics↗