Browsing by Author "Wright, R. Clay"

Now showing 1 - 11 of 11
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    Accelerating structure-function mapping using the ViVa webtool to mine natural variation
    Hamm, Morgan; Moss, Britney; Leydon, Alexander; Gala, Hardik; Lanctot, Amy; Ramos, Román; Klaeser, Hannah; Lemmex, Andrew; Zahler, Mollye; Nemhauser, Jennifer L.; Wright, R. Clay (Wiley, 2018-12-05)
    Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).
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    Advancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapy
    Wright, R. Clay; Khakhar, Arjun; Eshleman, James R.; Ostermeier, Marc (2014-01)
    While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
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    AssemblyTron: Flexible automation of DNA assembly with Opentrons OT-2 lab robots
    Bryant, John A., Jr.; Kellinger, Mason; Longmire, Cameron; Miller, Ryan; Wright, R. Clay (Oxford University Press, 2022-12-22)
    As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn approach to simultaneously learn how function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize, and algorithmically perform each step of the Design-Build-Test-Learn cycle. However, there are many fewer options for automating the Build step. Build typically involves DNA assembly, which remains manual, low throughput, and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron: an open-source python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies beginning from fragment amplification. We show that AssemblyTron can perform PCRs across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent in vivo assemblies with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent in vivo assembly also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs, and wastes associated with synthetic biology, which along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering.
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    Characterizing Auxin Response Circuits in Saccharomyces cerevisiae by Flow Cytometry.
    Pierre-Jerome, Edith; Wright, R. Clay; Nemhauser, Jennifer L. (2017-01)
    Recapitulation of the nuclear auxin response pathway in Saccharomyces cerevisiae (yeast) provides a means to functionally assay the contribution of individual signaling components to response dynamics. Here, we describe a time course assay for characterizing auxin response circuits using flow cytometry. This method allows for quantitative measurements of the dynamic response of up to 12 circuits (strains) at once. We also describe a steady-state assay and how to utilize an R package we developed to facilitate data analysis.
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    Insights into the Evolution and Function of Auxin Signaling F-Box Proteins in Arabidopsis thaliana Through Synthetic Analysis of Natural Variants.
    Wright, R. Clay; Zahler, Mollye L.; Gerben, Stacey R.; Nemhauser, Jennifer L. (2017-10)
    The evolution of complex body plans in land plants has been paralleled by gene duplication and divergence within nuclear auxin-signaling networks. A deep mechanistic understanding of auxin signaling proteins therefore may allow rational engineering of novel plant architectures. Toward that end, we analyzed natural variation in the auxin receptor F-box family of wild accessions of the reference plant Arabidopsis thaliana and used this information to populate a structure/function map. We employed a synthetic assay to identify natural hypermorphic F-box variants and then assayed auxin-associated phenotypes in accessions expressing these variants. To more directly measure the impact of the strongest variant in our synthetic assay on auxin sensitivity, we generated transgenic plants expressing this allele. Together, our findings link evolved sequence variation to altered molecular performance and auxin sensitivity. This approach demonstrates the potential for combining synthetic biology approaches with quantitative phenotypes to harness the wealth of available sequence information and guide future engineering efforts of diverse signaling pathways.
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    Modular, robust, and extendible multicellular circuit design in yeast
    Carignano, Alberto; Chen, Dai Hua; Mallory, Cannon; Wright, R. Clay; Seelig, Georg; Klavins, Eric (eLife Sciences, 2022-03-21)
    Division of labor between cells is ubiquitous in biology but the use of multicellular consortia for engineering applications is only beginning to be explored. A significant advantage of multicellular circuits is their potential to be modular with respect to composition but this claim has not yet been extensively tested using experiments and quantitative modeling. Here, we construct a library of 24 yeast strains capable of sending, receiving or responding to three molecular signals, characterize them experimentally and build quantitative models of their input-output relationships. We then compose these strains into two- and three-strain cascades as well as a four-strain bistable switch and show that experimentally measured consortia dynamics can be predicted from the models of the constituent parts. To further explore the achievable range of behaviors, we perform a fully automated computational search over all two-, three-, and four-strain consortia to identify combinations that realize target behaviors including logic gates, band-pass filters, and time pulses. Strain combinations that are predicted to map onto a target behavior are further computationally optimized and then experimentally tested. Experiments closely track computational predictions. The high reliability of these model descriptions further strengthens the feasibility and highlights the potential for distributed computing in synthetic biology.
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    New tangles in the auxin signaling web.
    Wright, R. Clay; Nemhauser, Jennifer L. (2015-01)
    Plants use auxin to relay critical information that shapes their growth and development. Auxin perception and transcriptional activation are mediated by the degradation of Aux/IAA repressor proteins. Degradation of Aux/IAAs relieves repression on Auxin Response Factors (ARFs), which bind DNA sequences called Auxin Response Elements (AuxREs). In most higher plant genomes, multiple paralogs exist for each part of the auxin nuclear signaling pathway. This potential combinatorial diversity in signaling pathways likely contributes to the myriad of context-specific responses to auxin. Recent structures of several domains from ARF proteins have exposed new modes of ARF dimerization, new models for ARF-AuxRE specificity, and the strong likelihood of larger order complexes formed by ARF and Aux/IAA homo- and heteromultimerization. Preliminary experiments support a role for these novel interactions in planta, further increasing the potential architectural complexity of this seemingly simple pathway.
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    Plant Synthetic Biology: Quantifying the Known Unknowns and Discovering the Unknown Unknowns.
    Wright, R. Clay; Nemhauser, Jennifer L. (2019-01-10)
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    A protein therapeutic modality founded on molecular regulation.
    Wright, Chapman M.; Wright, R. Clay; Eshleman, James R.; Ostermeier, Marc (2011-09-19)
    The exquisite specificity of proteins is a key feature driving their application to anticancer therapies. The therapeutic potential of another fundamental property of proteins, their ability to be regulated by molecular cues in their environment, is unknown. Here, we describe a synthetic biology strategy for designing protein therapeutics that autonomously activate a therapeutic function in response to a specific cancer marker of choice. We demonstrate this approach by creating a prodrug-activating enzyme that selectively kills human cancer cells that accumulate the marker hypoxia-inducible factor 1α. This property arises primarily through increased cellular accumulation of the enzyme in the presence of the marker. Our strategy offers a platform for the development of inherently selective protein therapeutics for cancer and other diseases.
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    Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration
    Gonzalez, Juan H.; Taylor, Joseph S.; Reed, Kelsey M.; Wright, R. Clay; Bargmann, Bastiaan O. R. (MDPI, 2021-10-23)
    Background: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. Methods: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. Results: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. Conclusions: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.
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    Vision, challenges and opportunities for a Plant Cell Atlas
    Jha, Suryatapa Ghosh; Borowsky, Alexander T.; Cole, Benjamin J.; Fahlgren, Noah; Farmer, Andrew; Huang, Shao-Shan Carol; Karia, Purva; Libault, Marc; Provart, Nicholas J.; Rice, Selena L.; Saura-Sanchez, Maite; Agarwal, Pinky; Ahkami, Amir H.; Anderton, Christopher R.; Briggs, Steven P.; Brophy, Jennifer An; Denolf, Peter; Di Costanzo, Luigi F.; Exposito-Alonso, Moises; Giacomello, Stefania; Gomez-Cano, Fabio; Kaufmann, Kerstin; Ko, Dae Kwani; Kumar, Sagar; Malkovskiy, Andrey; Nakayama, Naomi; Obata, Toshihiro; Otegui, Marisa S.; Palfalvi, Gergo; Quezada-Rodriguez, Elsa H.; Singh, Rajveer; Uhrig, R. Glen; Waese, Jamie; Van Wijk, Klaas; Wright, R. Clay; Ehrhardt, David W.; Birnbaum, Kenneth D.; Rhee, Seung Y. (eLife, 2021-09-07)
    With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.