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  • Synthetic Biology and Assembly Cloning  (70)
  • Natural Disasters
  • Oxford University Press  (112)
  • American Meteorological Society
  • Annual Reviews
  • 1
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    Trade Liberalization versus Climate Change Policy for Reducing Greenhouse Gas Emissions in Agriculture: Some Insights from Norway (2015)
    Oxford University Press
    Details
    Publication Date: 2015-08-11
    Description: Using a mathematical programming model of Norwegian agriculture, we explore interconnections between trade liberalization and reductions in greenhouse gas (GHG) emissions. We show that the Doha Round proposals for a new agreement on agriculture through the World Trade Organization would not generate significant reductions in emissions. Further trade liberalization would reduce emissions by cutting agricultural production but would not change production methods. Imposing a carbon tax would lead both to a reduction in output and the extensification of production. In contrast, if farmers are allowed to claim a credit for carbon sequestration the effect is to intensify agricultural production.
    Keywords: F18 - Trade and Environment, Q17 - Agriculture in International Trade, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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  • 2
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    Post-translational control of genetic circuits using Potyvirus proteases (2016)
    Oxford University Press
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    Publication Date: 2016-07-28
    Description: Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Temperature Changes, Household Consumption, and Internal Migration: Evidence from Tanzania (2016)
    Oxford University Press
    Details
    Publication Date: 2016-07-09
    Description: Large rural-urban wage gaps observed in many developing countries are suggestive of barriers to migration that keep potential migrants in rural areas. Using long panel data spanning nearly two decades, I study the extent to which migration rates are constrained by liquidity constraints in rural Tanzania. The analysis begins by quantifying the impact of weather variation on household welfare. The results show how household consumption co-moves with temperature, rendering households vulnerable to local weather events. These temperature-induced income shocks are then found to inhibit long-term migration among men, thus preventing them from tapping into the opportunities brought about by geographical mobility.
    Keywords: O12 - Microeconomic Analyses of Economic Development, O15 - Human Resources ; Human Development ; Income Distribution ; Migration, Q54 - Climate ; Natural Disasters ; Global Warming, R23 - Regional Migration ; Regional Labor Markets ; Population
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 4
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    An efficient strategy for TALEN-mediated genome engineering in Drosophila (2013)
    Oxford University Press
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    Publication Date: 2013-09-26
    Description: In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
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    High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases (2013)
    Oxford University Press
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    Publication Date: 2013-06-08
    Description: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ~40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 6
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    Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation (2013)
    Oxford University Press
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    Publication Date: 2013-06-08
    Description: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p -azido- l -phenylalanine (pAzF) or p -propargyloxy- l -phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA opt ) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
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    Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast (2015)
    Oxford University Press
    Details
    Publication Date: 2015-05-03
    Description: Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Local warming and violent conflict in North and South Sudan (2015)
    Oxford University Press
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    Publication Date: 2015-04-14
    Description: Our article contributes to the emerging micro-level strand of the literature on the link between local variations in weather shocks and conflicts by focusing on a pixel-level analysis for North and South Sudan between 1997 and 2009. Temperature anomalies are found to strongly affect the risk of conflict, whereas the risk is expected to magnify in a range of 24–31% in the future under a median scenario. Our analysis also sheds light on the competition over natural resources, in particular water, as the main driver of such relationship in a region where pastoralism constitutes the dominant livelihood.
    Keywords: D74 - Conflict ; Conflict Resolution ; Alliances, O13 - Agriculture ; Natural Resources ; Energy ; Environment ; Other Primary Products, Q54 - Climate ; Natural Disasters ; Global Warming, R11 - Regional Economic Activity: Growth, Development, and Changes
    Print ISSN: 1468-2702
    Electronic ISSN: 1468-2710
    Topics: Geography , Economics
    Published by Oxford University Press
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  • 9
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    Dealing with Uncertainty in Agent-Based Simulation: Farm-Level Modeling of Adaptation to Climate Change in Southwest Germany (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-18
    Description: Climate change will most likely confront agricultural producers with natural, economic, and political conditions that have not previously been observed and are largely uncertain. As a consequence, extrapolation from past data reaches its limits, and a process-based analysis of farmer adaptation is required. Simulation of changes in crop yields using crop growth models is a first step in that direction. However, changes in crop yields are only one pathway through which climate change affects agricultural production. A meaningful process-based analysis of farmer adaptation requires a whole-farm analysis at the farm level. We use a highly disaggregated mathematical programming model to analyze farm-level climate change adaptation for a mountainous area in southwest Germany. Regional-level results are obtained by simulating each full-time farm holding in the study area. We address parameter uncertainty and model underdetermination using a cautious calibration approach and a comprehensive uncertainty analysis. We deal with the resulting computational burden using efficient experimental designs and high-performance computing. We show that in our study area, shifted crop management time slots can have potentially significant effects on agricultural supply, incomes, and various policy objectives promoted under German and European environmental policy schemes. The simulated effects are robust against model uncertainty and underline the importance of a comprehensive assessment of climate change impacts beyond merely looking at crop yield changes. Our simulations demonstrate how farm-level models can contribute to a process-based analysis of climate change adaptation if they are embedded into a systematic framework for treating inherent model uncertainty.
    Keywords: C61 - Optimization Techniques ; Programming Models ; Dynamic Analysis, C63 - Computational Techniques, Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 10
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    A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-21
    Description: RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP -transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 11
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    Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 12
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    Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 13
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    Irrigation Decisions for Major West Coast Crops: Water Scarcity and Climatic Determinants (2015)
    Oxford University Press
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    Publication Date: 2015-12-13
    Description: This article uses the 2007 Farm and Ranch Irrigation Survey database developed by the U.S. Department of Agriculture to assess the impact of water scarcity and climate on irrigation decisions for producers of specialty crops, wheat, and forage crops. We estimate an irrigation management model for major crops in the West Coast (California, Oregon, and Washington), which includes a farm-level equation of irrigated share and crop-specific equations of technology adoption and water application rate (orchard/vineyard, vegetable, wheat, alfalfa, hay, and pasture). We find that economic and physical water scarcity, climate, and extreme weather, such as frost, extreme heat, and drought, significantly impact producers’ irrigation decisions. Producers use sprinkler technologies or additional water applications to mitigate risk of crop damage from extreme weather. Water application rates are least responsive to surface water cost or groundwater well depth for producers of orchard/vineyard. Water supply institutions influence producers’ irrigation decisions. Producers who receive water from federal agencies use higher water application rates and are less likely to adopt water-saving irrigation technologies for some crops. Institutional arrangements, including access to distinct water sources (surface or ground) and whether surface water cost is fee based, also affect the responsiveness of water application rates to changes in surface water cost. The analysis provides valuable information about how producers in irrigated agricultural production systems would respond and adapt to water pricing policies and climate change.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q16 - R&D ; Agricultural Technology ; Agricultural Extension Services, Q18 - Agricultural Policy ; Food Policy, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 14
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    An Introduction to the Green Paradox: The Unintended Consequences of Climate Policies (2015)
    Oxford University Press
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    Publication Date: 2015-07-18
    Description: How important is the Green Paradox? We address this question in three ways. First, we present a simple model explaining how announcing a future climate policy may increase carbon emissions today – the Green Paradox effect. This effect is a result of fossil fuel producers increasing their extraction today as a response to a reduction in future resource rents. Second, we examine the theoretical and empirical literature to assess whether green paradoxes are likely to occur, and if they are, whether they are big enough to be of concern for policy makers. We consider several factors that affect the existence of the green paradox, including long-term extraction costs, short-term extraction capacities, the mix of policy instruments, and potential spatial carbon leakage to countries that have no climate policy. We find that these and other factors can sometimes strengthen, but mostly weaken, the case for concern about the green paradox. Third, we identify the lessons the literature offers for policy makers. We argue that in designing climate policy, policy makers need to consider the supply side of the fossil fuel market.
    Keywords: H23 - Externalities ; Redistributive Effects ; Environmental Taxes and Subsidies, Q31 - Demand and Supply, Q38 - Government Policy, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
    Published by Oxford University Press
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  • 15
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    Introductory Comment-The Green Paradox: A Supply-Side View of the Climate Problem (2015)
    Oxford University Press
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    Publication Date: 2015-07-18
    Description: Why have policies aimed at reducing the demand for carbon not succeeded in slowing down global carbon extraction and CO 2 emissions, and why have carbon prices failed to increase over the last three decades? This comment argues that this is because of the Green Paradox, that is, the anticipation of sales by resource owners who try to preempt the destruction of their markets by green policies. Reviewing some of the conditions under which strong and weak versions of the Green Paradox may emerge, it is argued that there is little hope that green replacement technologies will impose hard price constraints that would keep long-run extraction within a fixed carbon budget and that, therefore, even strong versions of the paradox cannot easily be avoided.
    Keywords: O13 - Agriculture ; Natural Resources ; Energy ; Environment ; Other Primary Products, Q32 - Exhaustible Resources and Economic Development, Q54 - Climate ; Natural Disasters ; Global Warming, H23 - Externalities ; Redistributive Effects ; Environmental Taxes and Subsidies
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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  • 16
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    The Green Paradox in Open Economies: Lessons from Static and Dynamic Models (2015)
    Oxford University Press
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    Publication Date: 2015-07-18
    Description: This article examines how, in a world with incomplete coordination among countries, well-intentioned unilateral environmental policies may actually harm the global environment. This outcome is known as the "Green Paradox." The incentives for free-riding and the challenge of achieving an effective international environmental agreement are reviewed. I examine the various channels that lead to carbon leakage in static models of open economies, and report some simulation results. This is complemented by a review of the potential for Green Paradox outcomes in dynamic open-economy models in which forward-looking firms exploit an exhaustible resource. I show that border tax adjustments can lead to Green Paradox outcomes. I also discuss priorities for future research on environmental policies in a trading world that lacks a central enforcement agency.
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, Q42 - Alternative Energy Sources
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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  • 17
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    Reflections-Managing Uncertain Climates: Some Guidance for Policy Makers and Researchers (2015)
    Oxford University Press
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    Publication Date: 2015-07-18
    Description: Climate change—and, by extension, climate policy—is beset with unknowns and unknowables. This "Reflections" article presents an overview of approaches to managing climate uncertainties, in the hopes of providing guidance for current policy decisions as well as future research. We propose the following guidance for policy makers: Treat climate change as a risk management problem; recognize that benefit-cost analysis is only the first of many steps in deciding on optimal climate policy; in assessing abatement choices, use a discount rate that declines over time; recognize the importance of framing, evidence, and connecting the dots; reward modesty. We suggest the following questions for consideration by researchers: Can we improve forecasting? Can we improve the way we address nonlinearities and possible irreversibilities? What other (sub)disciplines merit a closer look? How can we create the right incentives for updating and expanding economic damage functions and climate-economy models? What alternative decision criteria merit further exploration? What does ‘not knowing’ tell us?
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, D81 - Criteria for Decision-Making under Risk and Uncertainty
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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  • 18
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    Blue light-mediated transcriptional activation and repression of gene expression in bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: 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.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 19
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    Drought Index Insurance for the Central Valley Project in California (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: A multi-year drought has taken a severe toll on the agricultural economy of California’s Central Valley. Index insurance is an instrument with the potential to protect water users from economic losses due to periodic water shortages. An index insurance product based on the Sacramento Index and adapted to the Central Valley Project water supply is proposed. To address the potential for intertemporal adverse selection, three product designs are suggested: (1) "early bird" insurance; (2) variable premium insurance; and (3) variable deductible insurance. The performance of the designs are assessed using loss functions from the Westlands Water District in the San Joaquin Valley.
    Keywords: Q14 - Agricultural Finance, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 20
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    CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: 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.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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  • 21
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    Robust, tunable genetic memory from protein sequestration combined with positive feedback (2015)
    Oxford University Press
    Details
    Publication Date: 2015-10-15
    Description: Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10 6 -fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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    Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches (2016)
    Oxford University Press
    Details
    Publication Date: 2016-06-03
    Description: We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong 70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Quality Matters More Than Quantity: Asymmetric Temperature Effects on Crop Yield and Quality Grade (2016)
    Oxford University Press
    Details
    Publication Date: 2016-07-09
    Description: Climate change affects agriculture by altering not only output quantity, but also crop quality. We quantify the economic impacts of climate change on agriculture through changes in both quantity and quality, where quality is measured by crop grades. Our model controls for methodological issues regarding sample selection, aggregation, phenology, and nonlinearity. The empirical application to Japanese rice production indicates that temperature effects are asymmetric: quantity is especially vulnerable to cold, whereas quality is vulnerable to extremely high temperature. Using these results, we simulate the effect of global warming, and we find that warming (a 3 °C increase) increases farm revenues by improving yield but decreases revenues as a result of deteriorating quality. The net effect is negative, suggesting that quality matters more than quantity. The negative effect, however, can be mitigated by shifting cultivation periods and/or regions. Overall, our results suggest that the estimated impacts of climate change and adaptation strategies could be severely misleading unless quality is considered.
    Keywords: L15 - Information and Product Quality ; Standardization and Compatibility, Q10 - General, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    The Effect of Weather-Induced Internal Migration on Local Labor Markets. Evidence from Uganda (2015)
    Oxford University Press
    Details
    Publication Date: 2015-05-21
    Description: Relying on census data collected in 2002 and historical weather data for Uganda, we estimate the impact of weather-induced internal migration on the probability for non-migrants living in the destination regions to be employed. Consistent with the prediction of a simple theoretical model, our results reveal a larger negative impact than the one documented for developed countries. They further show that this negative impact is significantly stronger in Ugandan regions with lower road density and therefore less conducive to capital mobility: a 10 percentage points increase in the net in-migration rate in these areas decreases the probability of being employed of non-migrants by more than 10 percentage points.
    Keywords: E24 - Employment ; Unemployment ; Wages ; Intergenerational Income Distribution, J21 - Labor Force and Employment, Size, and Structure, J61 - Geographic Labor Mobility ; Immigrant Workers, Q54 - Climate ; Natural Disasters ; Global Warming, R23 - Regional Migration ; Regional Labor Markets ; Population
    Print ISSN: 0258-6770
    Electronic ISSN: 1564-698X
    Topics: Economics
    Published by Oxford University Press
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    Optimal Directions for Directional Distance Functions: An Exploration of Potential Reductions of Greenhouse Gases (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-18
    Description: This article explores the reduction potential of greenhouse gases for major pollution-emitting countries of the world using nonparametric productivity measurement methods and directional distance functions. In contrast to the existing literature, we apply optimization methods to endogenously determine optimal directions for the efficiency analysis. These directions represent the compromise of output enhancement and emissions reduction. The results show that for reasonable directions the adoption of best practices would lead to sizable emission reductions in a range of approximately 20% compared with current levels.
    Keywords: C14 - Semiparametric and Nonparametric Methods, D24 - Production ; Cost ; Capital and Total Factor Productivity ; Capacity, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-21
    Description: We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of 〉60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of 〉150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Regulation of Natural Gas in the United States, Canada, and Europe: Prospects for a Low Carbon Fuel (2015)
    Oxford University Press
    Details
    Publication Date: 2015-01-29
    Description: The United States and Canada have seen a competitive and technological revolution in unconventional natural gas production in the 21 st Century—dramatically lowering the price of gas and displacing high-carbon coal with low-carbon gas for power generation. This gas revolution came from an earlier revolution in the regulation of gas pipelines, which ended the obstruction of gas markets by pipeline interests. Neither revolution has spread to Europe, where increasingly protectionist EU legislation has effectively blocked competitive pipeline entry and related gas markets. As a result, unconventional gas is untapped, coal displaces gas for power generation, and oil-linked gas prices have cost EU consumers a staggering $425 billion more than their US counterparts have paid since 2009 for about the same quantity of gas. Europe faces a serious institutional challenge to adopting the kind of pipeline regulation that facilitates the competitive flow of natural gas supplies and the accompanying lower carbon emissions. ( JEL : D23, K23, L14, L51, L95, N70, Q54)
    Keywords: D23 - Organizational Behavior ; Transaction Costs ; Property Rights, K23 - Regulated Industries and Administrative Law, L14 - Transactional Relationships ; Contracts and Reputation ; Networks, L51 - Economics of Regulation, L95 - Gas Utilities ; Pipelines ; Water Utilities, N70 - General, International, or Comparative, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    A Global Perspective on the Future of Natural Gas: Resources, Trade, and Climate Constraints (2015)
    Oxford University Press
    Details
    Publication Date: 2015-01-29
    Description: Natural gas plays an important role in the global energy system as an input to power generation, heating, and industry. This article identifies key drivers and uncertainties for natural gas markets in the coming decades. These include the availability of natural gas from conventional and unconventional sources, the role of international trade, and the impact of climate policies. We build on model-based research as well as an up-to-date survey of natural gas resource availability. We find that natural gas is an abundant fossil fuel and that the Asia-Pacific region will be most important in future global natural gas markets, especially under stringent international climate change mitigation. This means that an increasingly large share of future natural gas trade flows and infrastructure expansions will be directed to the Asia-Pacific region and that the role of liquefied natural gas will continue to increase globally. ( JEL : C61, L71, Q33, Q37, Q54)
    Keywords: C61 - Optimization Techniques ; Programming Models ; Dynamic Analysis, L71 - Mining, Extraction, and Refining: Hydrocarbon Fuels, Q33 - Resource Booms, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    Fiscal Responses after Catastrophes and the Enabling Role of Financial Development (2015)
    Oxford University Press
    Details
    Publication Date: 2015-01-29
    Description: Natural disasters may constitute a major shock to public finances and debt sustainability because of their impact on output and the need for government response with reconstruction and relief expenses. The question arises of whether governments can use financial development policy as the means to mitigate or insure against this negative fiscal impact. This paper uses a panel vector autoregressive model, estimated on annual data for high- and middle-income countries over 1975–2008, to study the role of debt market development and insurance penetration in enabling fiscal response after catastrophes. The authors find that countries with higher debt market development suffer smaller real consequences from disasters but that their deficits expand further following the mitigating fiscal response. Disasters in countries with high insurance penetration also experience smaller real consequences of disasters but without the need for further deficit expansions. From an ex-post perspective, the availability of insurance could offer the best mitigation approach against the real and fiscal consequences of disasters.
    Keywords: E32 - Business Fluctuations ; Cycles, H50 - General, H60 - General, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0258-6770
    Electronic ISSN: 1564-698X
    Topics: Economics
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    Distributional Implications of Climate Change in Rural India: A General Equilibrium Approach (2015)
    Oxford University Press
    Details
    Publication Date: 2015-07-10
    Description: We develop a general equilibrium framework, based on a specific-factors trade model, to quantify the medium-term household welfare impacts of global warming in rural India. Using an hedonic approach grounded in the theory combined with detailed microdata, we estimate that three decades of warming will reduce agricultural productivity in the range of 7%–13%, with the arid northwest of India especially hard hit. Our analysis shows that the proportional welfare cost of climate change is likely to be both modest and evenly distributed across percentiles of the per capita income distribution, but this latter conclusion emerges only when the flexibility of rural wages is taken into account.
    Keywords: Q17 - Agriculture in International Trade, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection (2012)
    Oxford University Press
    Details
    Publication Date: 2012-09-27
    Description: Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Alternative Metrics for Comparing Domestic Climate Change Mitigation Efforts and the Emerging International Climate Policy Architecture (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-03
    Description: The availability of practical mechanisms for comparing domestic efforts aimed at mitigating global climate change is important for the stability, equity, and efficiency of international climate agreements. We examine a variety of metrics that could be used to compare countries’ climate change mitigation efforts and illustrate their potential application to large developed and developing countries. Because there is no single, comprehensive, measurable metric that could be applied to all countries, we suggest using a set of indicators to characterize and compare mitigation effort, akin to using a set of economic statistics to indicate the health of the macroeconomy. Given the iterative pledge-and-review approach that is emerging in the current climate change negotiations, participation, commitment, and compliance could be enhanced if this set of indicators is able to show that all parties are doing their "fair share," both prospectively and retrospectively. The latter, in particular, highlights the need for a well-functioning policy surveillance regime. ( JEL : Q54, Q58, F55)
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, Q58 - Government Policy, F55 - International Institutional Arrangements
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    The Economics of Wine, Weather, and Climate Change (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-03
    Description: In this article, we provide an overview of the extensive literature on the impact of weather and climate on grapes and wine, with the goal of identifying how climate change is likely to affect their production. We first discuss the physical impact of weather on vine phenology (i.e., the timing of biological events such as bud break or flowering), berry composition, and yields. Then we examine the economic literature that measures the effects of temperature on wine quality, prices, costs, and profits and, based on this review, infer how climate change will affect these variables. We also describe what has been learned thus far about possible adaptation strategies for grape growers that would allow them to mitigate the economic effects of climate change. We conclude that climate change is likely to produce both winners and losers, with the winners being those located closer to the North and South Poles. There are also likely to be some substantial short-run costs as growers adapt to climate change. Nevertheless, wine making has survived through thousands of years of recorded history, a history that has included significant climate changes. ( JEL : Q13, Q18, Q54)
    Keywords: Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness, Q18 - Agricultural Policy ; Food Policy, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    The Impact of the European Union Emissions Trading Scheme on Regulated Firms: What Is the Evidence after Ten Years? (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-03
    Description: This article reviews the recent literature on ex post evaluation of the impacts of the European Union (EU) Emissions Trading Scheme (ETS) on regulated firms in the industrial and power sectors. We summarize the findings from original research papers concerning three broadly defined impacts: carbon dioxide emissions, economic performance and competitiveness, and innovation. We conclude by highlighting gaps in the current literature and suggesting priorities for future research on this landmark policy. ( JEL : Q52, Q54, Q58)
    Keywords: Q52 - Pollution Control Costs ; Distributional Effects ; Employment Effects, Q54 - Climate ; Natural Disasters ; Global Warming, Q58 - Government Policy
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    The European Union Emissions Trading System: Ten Years and Counting (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-03
    Description: This article provides an introduction to the European Union (EU) Emissions Trading System (ETS). First we describe the legislative development of the EU ETS, its evolution from free allocation to auctioning and centralized allocation rules, its relationship to the Kyoto Protocol and other trading systems, and its relationship to other EU climate and energy policies. This is followed by an assessment of the performance of the EU ETS, which focuses in particular on emissions, allowance prices, and the use of offsets. We conclude with a discussion of the current debate about the future of the EU ETS and proposals for changes to both the EU ETS and the climate policy environment in which it operates. ( JEL : Q54, Q58)
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, Q58 - Government Policy
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    The Climate Change Adaptation Literature (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-03
    Description: The December 2015 Conference of the Parties (COP) to the United Nations Framework Convention on Climate Change meetings in Paris are likely to yield a global agreement that will slow the world’s growth of greenhouse gas emissions, but this agreement is unlikely to guarantee a decline in global emissions in the near future. Given this reality, climate change adaptation is an increasingly important topic for discussion and study. Although much research has focused on the macroeconomic relationship between economic growth and temperature at the national and/or annual level, microeconomic analysis also offers valuable insights. This Reflections discusses recent work on household and firm responses to three climate change challenges: increased summer heat, higher food prices, and increased natural disaster risk. ( JEL : Q54)
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    Synthesis and cell-free cloning of DNA libraries using programmable microfluidics (2016)
    Oxford University Press
    Details
    Publication Date: 2016-03-01
    Description: Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    A gene network engineering platform for lactic acid bacteria (2016)
    Oxford University Press
    Details
    Publication Date: 2016-03-01
    Description: Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Endogenous farm-type selection, endogenous irrigation, and spatial effects in Ricardian models of climate change (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-20
    Description: In the estimation of Ricardian models the endogeneity of adaptation measures is typically ignored. In this article we propose a new estimation strategy that explicitly recognises the endogeneity of the farm type and irrigation to climate. Based on the latest census data on over 270,000 farms in Germany, we estimate a cross-sectional, spatial-IV model that decomposes the effects of climate on farm profitability into direct (unmediated) and indirect (mediated by the variables that reflect adaptation). Our results show that neglecting the endogenous nature of adaptation measures may substantially bias the magnitude of the total effect of climate on farm profitability.
    Keywords: C21 - Cross-Sectional Models ; Spatial Models ; Treatment Effect Models, C25 - Discrete Regression and Qualitative Choice Models, C36- Instrumental Variables (IV) Estimation, Q18 - Agricultural Policy ; Food Policy, Q51 - Valuation of Environmental Effects, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor (2015)
    Oxford University Press
    Details
    Publication Date: 2015-12-02
    Description: Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Responsiveness of Crop Yield and Acreage to Prices and Climate (2015)
    Oxford University Press
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    Publication Date: 2015-12-13
    Description: We investigate the effect of crop price and climate variables on rainfed corn and soybean yields and acreage in the United States using a large panel dataset for the 1977–2007 period. Instrumental variables are used to control for endogeneity of prices in yield and acreage regressions, while allowing for spatially auto-correlated errors. We find that an increase in corn price has a statistically significant positive impact on corn yield, but the effect of soybean price on soybean yields is not statistically significant. The estimated price elasticities of corn yield and acreage are 0.23 and 0.45, respectively. Of the increase in corn supply caused by an increase in corn price, we find that 33.8% is due to price-induced yield enhancement and 66.2% is due to price-induced acreage expansion. We also find that the impact of climate change on corn production ranges from $-$ 7% to $-$ 41% and on soybean ranges from $-$ 8% to $-$ 45%, depending on the climate change scenarios, time horizon, and global climate models used to predict climate change. We show that the aggregate net impact of omitting price variables is an overestimation of the effect of climate change on corn yield by up to 9% and on soybean yield by up to 15%.
    Keywords: Q11 - Aggregate Supply and Demand Analysis ; Prices, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter (2012)
    Oxford University Press
    Details
    Publication Date: 2012-06-06
    Description: A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    SLiCE: a novel bacterial cell extract-based DNA cloning method (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-24
    Description: We describe a novel cloning method termed SLiCE (Seamless L i gation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-24
    Description: Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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  • 45
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    A transposase strategy for creating libraries of circularly permuted proteins (2012)
    Oxford University Press
    Details
    Publication Date: 2012-05-13
    Description: A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Looking into the future of agriculture in a changing climate (2011)
    Oxford University Press
    Details
    Publication Date: 2011-11-24
    Description: Looking into the future of agriculture raises three challenging questions: How can agriculture deal with an uncertain future? How do local vulnerabilities and global disparities respond to this uncertain future? How should we prioritise adaptation to overcome the resulting future risks? This paper analyses the broad question of how climate change science may provide some insights into these issues. The data provided for the analysis are the product of our new research on global impacts of climate change in agriculture. The questions are analysed across world regions to provide some thoughts on policy development.
    Keywords: N50 - General, International, or Comparative, Q18 - Agricultural Policy ; Food Policy, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency (2012)
    Oxford University Press
    Details
    Publication Date: 2012-02-28
    Description: Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli . This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Population, Poverty, and Climate Change (2014)
    Oxford University Press
    Details
    Publication Date: 2014-01-22
    Description: This literature review focuses on the relationships between population, poverty, and climate change. Developed countries are largely responsible for global warming, but the brunt of the fallout will be borne by developing countries in forms such as lower agricultural output, poorer health, and more frequent natural disasters. Although carbon emissions per capita have leveled off in developed countries, they are projected to rise rapidly in developing countries because of economic growth and population growth. Unfortunately, the latter will rise most notably in the poorest countries, combining with climate change to slow poverty reduction. These countries have many incentives to lower fertility. Previous studies indicate that in high fertility settings, fertility decline facilitates economic growth and poverty reduction. It also reduces the pressure on livelihoods and frees resources that can be used to cope with climate change. Moreover, slowing population growth helps avert some of the projected global warming, which will benefit the poorest countries far more than it will benefit developed countries that lie at higher latitudes and/or have more resources to cope with climate change. Natural experiments indicate that family-planning programs are effective and highly pro-poor in their impact. While the rest of the world wrestles with the complexities of reducing emissions, the poorest countries will benefit from simple programs to lower fertility.
    Keywords: Q56 - Environment and Development ; Environment and Trade ; Sustainability ; Environmental Accounting ; , Q54 - Climate ; Natural Disasters ; Global Warming, J13 - Fertility ; Family Planning ; Child Care ; Children ; Youth, J18 - Public Policy
    Print ISSN: 0257-3032
    Electronic ISSN: 1564-6971
    Topics: Economics
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    DICE, an efficient system for iterative genomic editing in human pluripotent stem cells (2014)
    Oxford University Press
    Details
    Publication Date: 2014-03-13
    Description: To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11 , located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a ‘landing pad’ cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson’s disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.
    Keywords: Synthetic Biology and Assembly Cloning
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    Improved seamless mutagenesis by recombineering using ccdB for counterselection (2014)
    Oxford University Press
    Details
    Publication Date: 2014-03-13
    Description: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli , usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.
    Keywords: Synthetic Biology and Assembly Cloning
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    Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli (2013)
    Oxford University Press
    Details
    Publication Date: 2013-12-07
    Description: The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc R ) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc R . A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.
    Keywords: Synthetic Biology and Assembly Cloning
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    High-throughput, cost-effective verification of structural DNA assembly (2014)
    Oxford University Press
    Details
    Publication Date: 2014-02-28
    Description: DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (~1 kb), pathway engineering (~10 kb) or synthetic genomes (〉100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at 〈$1 per sample.
    Keywords: Synthetic Biology and Assembly Cloning
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    Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination (2014)
    Oxford University Press
    Details
    Publication Date: 2014-02-28
    Description: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP / attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 54
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    Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-03
    Description: A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z 3 EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z 3 EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z 3 EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
    Keywords: Synthetic Biology and Assembly Cloning
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    Information Manipulation and Climate Agreements (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-05
    Description: It appears that news media and some pro-environmental organizations have the tendency to accentuate or even exaggerate the damage caused by climate change. This article provides a rationale for this tendency by using a modified International Environmental Agreement (IEA) model with asymmetric information. We find that the information manipulation has an instrumental value, as it ex post induces more countries to participate in an IEA, which will eventually enhance global welfare. From the ex ante perspective, however, the impact that manipulating information has on the level of participation in an IEA and on welfare is ambiguous.
    Keywords: D82 - Asymmetric and Private Information, L82 - Entertainment ; Media, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    An exogenous chloroplast genome for complex sequence manipulation in algae (2012)
    Oxford University Press
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    Publication Date: 2012-03-29
    Description: We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii . Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus . Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.
    Keywords: Synthetic Biology and Assembly Cloning
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    A versatile element for gene addition in bacterial chromosomes (2012)
    Oxford University Press
    Details
    Publication Date: 2012-02-17
    Description: The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp . Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector.
    Keywords: Synthetic Biology and Assembly Cloning
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    Error correction of microchip synthesized genes using Surveyor nuclease (2012)
    Oxford University Press
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    Publication Date: 2012-02-17
    Description: The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'-〉5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by 〉16-fold, yielding a final error rate of ~1 in 8700 bp.
    Keywords: Synthetic Biology and Assembly Cloning
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    Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-27
    Description: The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.
    Keywords: Synthetic Biology and Assembly Cloning
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    Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria (2014)
    Oxford University Press
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    Publication Date: 2014-09-27
    Description: Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.
    Keywords: Synthetic Biology and Assembly Cloning
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    A platform for rapid prototyping of synthetic gene networks in mammalian cells (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-28
    Description: Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.
    Keywords: Synthetic Biology and Assembly Cloning
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    Customized optimization of metabolic pathways by combinatorial transcriptional engineering (2012)
    Oxford University Press
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    Publication Date: 2012-10-10
    Description: A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named ‘customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)’ for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.
    Keywords: Synthetic Biology and Assembly Cloning
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    'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules (2012)
    Oxford University Press
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    Publication Date: 2012-10-10
    Description: We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.
    Keywords: Synthetic Biology and Assembly Cloning
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    Adaptation to climate change in Sub-Saharan agriculture: assessing the evidence and rethinking the drivers (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-03
    Description: In this paper, after a review of the evolution of the literature on climate change economics in agriculture, I present some evidence of the impact of different moments of the distribution of rainfall on farmers risk aversion. It is found that while more rainfall is negatively associated with the probability of observing risk aversion, rainfall variability is positively correlated. This result highlights an important behavioural dimension of climatic factors.
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, Q56 - Environment and Development ; Environment and Trade ; Sustainability ; Environmental Accounting
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Rapid, modular and reliable construction of complex mammalian gene circuits (2013)
    Oxford University Press
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    Publication Date: 2013-09-06
    Description: We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.
    Keywords: Synthetic Biology and Assembly Cloning
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    Multiplexing clonality: combining RGB marking and genetic barcoding (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-15
    Description: RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-15
    Description: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for 〉100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo .
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    PaperClip: rapid multi-part DNA assembly from existing libraries (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-12
    Description: Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Antagonistic control of a dual-input mammalian gene switch by food additives (2014)
    Oxford University Press
    Details
    Publication Date: 2014-08-15
    Description: Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni , which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Multiplex pairwise assembly of array-derived DNA oligonucleotides (2016)
    Oxford University Press
    Details
    Publication Date: 2016-03-19
    Description: While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Dynamic Adjustment in US Agriculture under Climate Change (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-24
    Description: We construct a stochastic dynamic dual model to investigate the structural adjustment of two aggregate output and three aggregate input categories in US agriculture under stochastic climatic change. More than a century of national annual data (1910–2011) is used in the empirical analysis. No constraints on asset fixity are imposed. Results indicate that, with rational expectations, both output categories as well as all input categories exhibit quasi-fixity in response to market change and stochastic climate change. Crops adjust more than twice as fast as livestock—49% versus 20% of the way toward their long-run equilibrium in one year. Fertilizer adjusts most rapidly toward equilibrium levels (88% in one year), and capital adjusts most slowly (5% in one year). Labor oscillates rather than converging smoothly toward equilibrium; its distance from equilibrium is the same as if it adjusted 59% of the way toward its optimal level in one year. Failing to anticipate climate change dramatically slows the estimated rate of adjustment for two netputs and modestly speeds the rate for two others, thus likely increasing overall adjustment costs. Failing to account for uncertainty in anticipated climate change has little impact on adjustment rates.
    Keywords: Q11 - Aggregate Supply and Demand Analysis ; Prices, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Agriculture Afforestation for Carbon Sequestration Under Carbon Markets in the United States: Leakage Behavior from Regional Allowance Programs (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-10
    Description: This study quantifies how leakage behavior from afforesting agricultural land affects the intensification of agricultural production. In particular, we examine the leakage percentage from carbon offset allowance at specific southern regions in the United States as a part of a carbon market. We use the Forest and Agriculture Sector Optimization Model-Greenhouse Gases model to examine responses between sectors as part of the regional afforestation policy analysis. Regional characteristics and a policy's time frame are found to play important roles in achieving net gains, in terms of greenhouse gases stored, from such regional policies. In some cases, however, leakage greater than 100% is evident.
    Keywords: Q23 - Forestry, Q54 - Climate ; Natural Disasters ; Global Warming, R14 - Land Use Patterns
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution (2012)
    Oxford University Press
    Details
    Publication Date: 2012-12-14
    Description: Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K d ) 〈10 pM, representing a ~200-fold improvement in binding affinity over the monomeric aptamers and a ~15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Synthetic mammalian trigger-controlled bipartite transcription factors (2013)
    Oxford University Press
    Details
    Publication Date: 2013-07-16
    Description: Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), -butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and -butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    New Uses of Old Tools? Greenhouse Gas Mitigation with Agriculture Sector Policies (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-22
    Description: Following the failure of legislative proposals for a multi-sector greenhouse gas (GHG) cap-and-trade policy, the shift in focus to energy sector policies ignores the perhaps substantial potential for GHG mitigation from agriculture/forestry. We review estimates of the current U.S. agriculture sector contribution to GHG mitigation from a portfolio of existing sector policies in bioenergy, conservation, and research and development to compare accomplishments across programs. We then consider what opportunities and challenges may exist for increasing sector GHG mitigation by retargeting and/or expanding current programs—or for bioenergy-related mitigation, implementing proposed new programs—to serve as an alternative to cap-and-trade.
    Keywords: Q16 - R&D ; Agricultural Technology ; Agricultural Extension Services, Q42 - Alternative Energy Sources, Q54 - Climate ; Natural Disasters ; Global Warming, Q58 - Government Policy
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Cutting Carbon, Take Two: A Brief Guide to Federal Electricity-Sector Climate Policy without Cap-and-Trade (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-22
    Description: This paper explores federal policies, other than a carbon price, for reducing emissions from the electric power sector. These policies fall into two major categories: policies that encourage the development of non- or low-emitting energy sources, and regulatory policies under existing legal authority (primarily the Clean Air Act). The paper provides an overview of policy options and a few concrete proposals, along with a summary of insights from economists on their advantages and disadvantages. Economists generally disfavor investment subsidies, but comparing other policy options, including regulatory approaches, technology mandates, and production subsidies, is complex. Excluding existing clean generation from incentive policies is tempting but can lead to perverse outcomes.
    Keywords: L94 - Electric Utilities, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    A part toolbox to tune genetic expression in Bacillus subtilis (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-03
    Description: 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 .
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Climate change and regulation of nitrogen loads under moral hazard (2014)
    Oxford University Press
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    Publication Date: 2014-03-07
    Description: Within the European Union, it is agreed that watershed-based management of water quality problems is more efficient than centralised arrangements. In this study, a mechanism for allocating international funds to watershed authorities for nitrogen abatement in the presence of moral hazard is investigated. The results show that when there is a risk of climate change, the cost of moral hazard to the international funding agency can be high if there is a moderate likelihood of climate change and the watershed authority is guaranteed a high minimum compensation.
    Keywords: Q53 - Air Pollution ; Water Pollution ; Noise ; Hazardous Waste ; Solid Waste ; Recycling, Q54 - Climate ; Natural Disasters ; Global Warming, Q58 - Government Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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    Potential Effects of Climate Change on the Productivity of U.S. Dairies (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-23
    Description: In the United States, climate change is likely to increase average daily temperatures and the frequency of heat waves, which can reduce meat and milk production in animals. Methods that livestock producers use to mitigate thermal stress—including modifications to animal management or housing—tend to increase production costs. We use operation-level economic data coupled with finely-scaled climate data to estimate how the local thermal environment affects the technical efficiency of dairies across the United States. We then use this information to estimate the possible decline in milk production in 2030 resulting from climate change-induced heat stress under the simplifying assumptions that the production technology, location of production, and other factors are held constant. For four climate model scenarios, the results indicate modest heat-stress-related production declines by 2030, with the largest declines occurring in the southern states.
    Keywords: D24 - Production ; Cost ; Capital and Total Factor Productivity ; Capacity, Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Extreme Weather and Civil War: Does Drought Fuel Conflict in Somalia through Livestock Price Shocks? (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-23
    Description: A growing body of evidence shows a causal relationship between extreme weather events and civil conflict incidence at the global level. We find that this causality is also valid for droughts and local violent conflicts in a within-country setting over a short time frame in the case of Somalia. We estimate that a one standard deviation increase in drought intensity and length raises the likelihood of conflict by 62%. We also find that drought affects conflict through livestock price changes, establishing livestock markets as the primary channel of transmission in Somalia.
    Keywords: D74 - Conflict ; Conflict Resolution ; Alliances, O12 - Microeconomic Analyses of Economic Development, Q11 - Aggregate Supply and Demand Analysis ; Prices, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Solar Geoengineering's Brave New World: Thoughts on the Governance of an Unprecedented Technology (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-23
    Description: Due to the failure of international efforts to limit atmospheric concentrations of greenhouse gases, consideration is now being given to solar geoengineering—a deliberate intervention to limit global warming without altering the atmospheric concentration of greenhouse gases. In contrast to emission cuts, geoengineering is expected to be cheap, quick to lower temperature, and feasible through the use of a single intervention. However, geoengineering is an imperfect substitute for emission reductions and will likely have undesirable side effects, only some of which can be anticipated before geoengineering is deployed. Most importantly, because geoengineering can be undertaken unilaterally, it creates issues of governance: Who gets to decide if, when, and how geoengineering should be attempted? This article provides an introduction to the key issues surrounding the governance of this unprecedented technology. ( JEL : Q54, F53, K33)
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming, F53 - International Agreements and Observance ; International Organizations, K33 - International Law
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    Climate Engineering: Economic Considerations and Research Challenges (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-23
    Description: Climate engineering measures are designed to either reduce atmospheric carbon concentration (by growing trees or spreading iron in the ocean, for example) or directly influence the radiation reaching or leaving the earth (by injecting sulfur into the stratosphere or modifying cloud formations, for example) to compensate for greenhouse gas–induced warming. The former measures are termed carbon dioxide removal (CDR), which we characterize as a low-leverage causative approach, and the latter are termed radiation management (RM), which we characterize as a high-leverage symptomatic approach. There are similarities between CDR and emission control. Accordingly, benefit-cost analysis can be used to assess certain CDR measures. By contrast, high-leverage RM represents a genuinely new option in the climate change response portfolio, at first glance promising insurance against fat-tail climate change risks. However, the persistent intrinsic uncertainties of RM suggest that any cautious climate risk management approach should consider RM as a complement to (rather than a substitute for) emission control at best. Moreover, the complexity of the earth system imposes major limitations on the ability of research to reduce these uncertainties. Thus we argue that a research strategy is needed that focuses on increasing our basic understanding of the earth system and conducting comprehensive assessments of the risk(s) associated with both climate change and the deployment of climate engineering. ( JEL : Q52, Q54, Q55)
    Keywords: Q52 - Pollution Control Costs ; Distributional Effects ; Employment Effects, Q54 - Climate ; Natural Disasters ; Global Warming, Q55 - Technological Innovation
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    Climate Policy: Science, Economics, and Extremes (2014)
    Oxford University Press
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    Publication Date: 2014-07-23
    Description: Climate scientists, and natural scientists more generally, believe that climate change is a major, perhaps the most important, problem facing humankind this century, and that it is increasingly linked to extreme weather events. However, the impression one gets from much of the economic literature, particularly simulations from integrated assessment models used in policy analysis, is that the potential impacts of climate change are not large enough to warrant aggressive mitigation efforts in the near term. Although these models represent an important step in the needed interdisciplinary analysis of climate change by elucidating the links between climate and economy, we argue that they grossly underestimate potential impacts and associated damages because they (and the related policy analyses) fail to adequately capture extreme conditions, catastrophic events, and tipping points that trigger irreversible changes in the climate system, as well as impacts on the natural environment that cannot be monetized. Because the most severe impacts are expected in the later years of this century and beyond, discounting is crucial, and we argue that the appropriate rate is well below market rates. Moreover, we show that in the uniquely long period relevant to climate policy, the irreversibility of climate changes and impacts is more serious than the irreversibility of proposed mitigation measures. We conclude that an aggressive mitigation policy is warranted, one that holds further increases in global mean temperature to the scientific consensus on what is required to avoid the worst impacts, and that such a policy can be achieved at a cost that is well below potential damages. ( JEL : Q54)
    Keywords: Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
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    A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers (2012)
    Oxford University Press
    Details
    Publication Date: 2012-08-08
    Description: Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye–dye and dye–DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.
    Keywords: Synthetic Biology and Assembly Cloning
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    Electronic ISSN: 1362-4962
    Topics: Biology
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    A versatile cis-blocking and trans-activation strategy for ribozyme characterization (2013)
    Oxford University Press
    Details
    Publication Date: 2013-01-20
    Description: Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity (2012)
    Oxford University Press
    Details
    Publication Date: 2012-11-04
    Description: Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10 6 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system (2013)
    Oxford University Press
    Details
    Publication Date: 2013-08-28
    Description: The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    A modular strategy for engineering orthogonal chimeric RNA transcription regulators (2013)
    Oxford University Press
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    Publication Date: 2013-08-28
    Description: Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli , we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 x 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Modular assembly of transposon integratable multigene vectors using RecWay assembly (2013)
    Oxford University Press
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    Publication Date: 2013-04-23
    Description: Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac ( PB ) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro , with up to 70-fold higher gene expression compared with analogous uninsulated vectors . RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly (2013)
    Oxford University Press
    Details
    Publication Date: 2013-04-23
    Description: Techniques for assembly of designed DNA sequences are important for synthetic biology. So far, a few methods have been developed towards high-throughput seamless DNA assembly in vitro , including both the homologous sequences-based system and the type IIS-mediated system. Here, we describe a novel method designated ‘MASTER Ligation’, by which multiple DNA sequences can be seamlessly assembled through a simple and sequence-independent hierarchical procedure. The key restriction endonuclease used, MspJI, shares both type IIM and type IIS properties; thus, it only recognizes the methylation-specific 4-bp sites, m CNNR (R = A or G), and cuts DNA outside of the recognition sequences. This method was tested via successful assembly of either multiple polymerase chain reaction amplicons or restriction fragments of the actinorhodin biosynthetic cluster of Streptomyces coelicolor (~29 kb), which was further heterologously expressed in a fast-growing and moderately thermophilic strain, Streptomyces sp. 4F.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Coordinated induction of multi-gene pathways in Saccharomyces cerevisiae (2013)
    Oxford University Press
    Details
    Publication Date: 2013-02-20
    Description: Bacterial operons are nature’s tool for regulating and coordinating multi-gene expression in prokaryotes. They are also a gene architecture commonly used in the biosynthesis of many pharmaceutically important compounds and industrially useful chemicals. Despite being an important eukaryotic production host, Saccharomyces cerevisiae has never had such gene architecture. Here, we report the development of a system to assemble and regulate a multi-gene pathway in S. cerevisiae . Full pathways can be constructed using pre-made parts from a plasmid toolbox. Subsequently, through the use of a yeast strain containing a stably integrated gene switch, the assembled pathway can be regulated using a readily available and inexpensive compound—estradiol—with extremely high sensitivity (10 nM). To demonstrate the use of the system, we assembled the five-gene zeaxanthin biosynthetic pathway in a single step and showed the ligand-dependent coordinated expression of all five genes as well as the tightly regulated production of zeaxanthin. Compared with a previously reported constitutive zeaxanthin pathway, our inducible pathway was shown to have 50-fold higher production level.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases (2013)
    Oxford University Press
    Details
    Publication Date: 2013-02-20
    Description: Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo , demonstrating that stitching yields ZFAs with robust recognition properties.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Measurement and modeling of intrinsic transcription terminators (2013)
    Oxford University Press
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    Publication Date: 2013-05-04
    Description: The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ~800-fold dynamic range within Escherichia coli . We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies ( r = 0.9, n = 31) better than models trained on all terminators ( r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation (2013)
    Oxford University Press
    Details
    Publication Date: 2013-06-28
    Description: Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l -lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l -lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l -lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum , the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-02
    Description: The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11 , were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination (2013)
    Oxford University Press
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    Publication Date: 2013-11-02
    Description: Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-02
    Description: The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (~10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems (2013)
    Oxford University Press
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    Publication Date: 2013-04-14
    Description: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.
    Keywords: Synthetic Biology and Assembly Cloning
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    Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish (2013)
    Oxford University Press
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    Publication Date: 2013-08-09
    Description: Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Reflections: Uncertainty and Decision Making in Climate Change Economics (2014)
    Oxford University Press
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    Publication Date: 2014-01-28
    Description: Uncertainty is intrinsic to climate change: we know that the climate is changing but not precisely how fast or in what ways. Nor do we understand fully the social and economic consequences of these changes or the options that will be available for reducing climate change. Furthermore, the uncertainty about these issues is not readily quantified in probabilistic terms: we are facing deep uncertainty rather than known risks. We argue that this may render the classical expected utility framework for decision making under uncertainty of limited value for informing climate policy. We review the sources of uncertainty about all aspects of climate change, separate these into scientific and socioeconomic components, and examine their relative importance. Then we review decision-making frameworks that may be more appropriate in the absence of unique probabilities including nonprobabilistic approaches and those based on multiple priors, and we discuss their application in the context of climate change economics. ( JEL : D81, Q54)
    Keywords: D81 - Criteria for Decision-Making under Risk and Uncertainty, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 1750-6816
    Electronic ISSN: 1750-6824
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Political Science , Economics
    Published by Oxford University Press
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