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  • Cooperatives  (74)
  • Synthetic Biology and Assembly Cloning  (70)
  • Oxford University Press  (144)
  • American Physical Society
  • Blackwell Publishing Ltd
  • Public Library of Science
  • 1
    Publikationsdatum: 2017-01-10
    Beschreibung: Despite the extensive use of  Saccharomyces cerevisiae  as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    Publikationsdatum: 2017-01-05
    Beschreibung: Commodity-equity return co-movements rose dramatically during the Great Recession. This development took place following what has been dubbed the "financialization" of commodity markets. We first document changes since 1995 in the relative importance of financial institutions’ activity in agricultural futures markets. We then use a structural vector autoregression (VAR) model to ascertain the role of that activity in explaining correlations between weekly grain, livestock, and equity returns from 1995–2015. We provide robust evidence that, accounting for shocks that are idiosyncratic to agricultural markets, world business cycle shocks have a substantial and long-lasting impact on the latter’s co-movements with financial markets. In contrast, changes in the intensity of financial speculation have an impact on cross-market return linkages that is shorter-lived and not statistically significant in all model specifications.
    Schlagwort(e): G12 - Asset Pricing ; Trading volume ; Bond Interest Rates, G13 - Contingent Pricing ; Futures Pricing, Q11 - Aggregate Supply and Demand Analysis ; Prices, Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness
    Print ISSN: 0002-9092
    Digitale ISSN: 1467-8276
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft , Wirtschaftswissenschaften
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
    Publikationsdatum: 2017-01-05
    Beschreibung: This article considers changes in the perception and avoidance behavior of food risks over time. In Japan, consumers’ concerns about the risks of radiation exposure via food consumption rose rapidly after the accident at the Fukushima Daiichi Nuclear Power Station in 2011. We conducted choice experiment surveys three months, seven months, and eleven months after the Fukushima accident and simulated the conditional means of willingness to pay (WTP) for avoiding radiation exposure via food consumption in each survey period. The results show that although the characteristics of changes in averting behaviors vary depending on the type of food, the heterogeneity in WTP values for avoiding food risk tends to reduce (broaden) when their magnitude decreases (increases).
    Schlagwort(e): Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness, Q18 - Agricultural Policy ; Food Policy, Q51 - Valuation of Environmental Effects
    Print ISSN: 0002-9092
    Digitale ISSN: 1467-8276
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft , Wirtschaftswissenschaften
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 4
    Publikationsdatum: 2016-12-07
    Beschreibung: The Agricultural Act of 2014 replaced dairy product price supports and countercyclical income support payments with the Margin Protection Program for Dairy Producers. Using farm-level data, producer decisions and aggregate policy costs under a variety of risk environments and policy design alternatives are simulated. Fixed premium rates may result in budget outlays that are substantially higher than for equivalent variable-rate insurance subsidized at levels observed in revenue-based crop insurance policies. Due to the absence of adjusted gross income or production eligibility constraints, a significant portion of benefits may accrue to a small share of large dairy farms.
    Schlagwort(e): Q11 - Aggregate Supply and Demand Analysis ; Prices, Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness
    Print ISSN: 2040-5790
    Digitale ISSN: 2040-5804
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft , Wirtschaftswissenschaften
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 5
    Publikationsdatum: 2016-12-01
    Beschreibung: Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain ( Rs LOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of Rs LOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, Rs LOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 6
    Publikationsdatum: 2016-10-08
    Beschreibung: A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 7
    Publikationsdatum: 2016-10-08
    Beschreibung: Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 8
    Publikationsdatum: 2016-09-03
    Beschreibung: Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli , such libraries are lacking for the Gram-positive model Bacillus subtilis , a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis . We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ~14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis .
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 9
    Publikationsdatum: 2016-08-20
    Beschreibung: Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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  • 10
    Publikationsdatum: 2016-08-20
    Beschreibung: Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA ( Cas 9-facilitated H omologous R ecombination A ssembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo ; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 ( M inimal G enome of Escherichia coli ) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.
    Schlagwort(e): Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Digitale ISSN: 1362-4962
    Thema: Biologie
    Publiziert von Oxford University Press
    Standort Signatur Erwartet Verfügbarkeit
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