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  • 1
    Publication Date: 2015-08-11
    Description: Existing economic analysis of corn stover as an energy feedstock has not considered potential changes in land use associated with different stover prices. We estimate the response of corn stover supply density to its price driven by changes in land use and examine its implications for a processing plant's pricing strategy and marginal cost, as well as associated changes in soil erosion. We find that plants will exploit the intensive margin as well as the extensive margin to secure additional amounts of stover. Our results show, counterintuitively, that a market for stover may result in lower soil erosion due to reallocations of land to continuous corn with removal, which, combined with no-till farming, results in lower soil erosion than the baseline without stover removal. Also contrary to expectations, using cover crops with stover removal may result in higher soil erosion due to land use changes within the fuel shed associated with optimal pricing.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q42 - Alternative Energy Sources
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 2
    Publication Date: 2015-09-19
    Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.
    Keywords: Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
    Publication Date: 2015-08-29
    Description: Data on biological mechanisms of aging are mostly obtained from cross-sectional study designs. An inherent disadvantage of this design is that inter-individual differences can mask small but biologically significant age-dependent changes. A serially sampled design (same individual at different time points) would overcome this problem but is often limited by the relatively small numbers of available paired samples and the statistics being used. To overcome these limitations, we have developed a new vector-based approach, termed three-component analysis, which incorporates temporal distance, signal intensity and variance into one single score for gene ranking and is combined with gene set enrichment analysis. We tested our method on a unique age-based sample set of human skin fibroblasts and combined genome-wide transcription, DNA methylation and histone methylation (H3K4me3 and H3K27me3) data. Importantly, our method can now for the first time demonstrate a clear age-dependent decrease in expression of genes coding for proteins involved in translation and ribosome function. Using analogies with data from lower organisms, we propose a model where age-dependent down-regulation of protein translation-related components contributes to extend human lifespan.
    Keywords: Genomics
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    Topics: Biology
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  • 4
    Publication Date: 2015-05-29
    Description: A major challenge in the field of shotgun metagenomics is the accurate identification of organisms present within a microbial community, based on classification of short sequence reads. Though existing microbial community profiling methods have attempted to rapidly classify the millions of reads output from modern sequencers, the combination of incomplete databases, similarity among otherwise divergent genomes, errors and biases in sequencing technologies, and the large volumes of sequencing data required for metagenome sequencing has led to unacceptably high false discovery rates (FDR). Here, we present the application of a novel, gene-independent and signature-based metagenomic taxonomic profiling method with significantly and consistently smaller FDR than any other available method. Our algorithm circumvents false positives using a series of non-redundant signature databases and examines G enomic O rigins T hrough T axonomic CHA llenge (GOTTCHA). GOTTCHA was tested and validated on 20 synthetic and mock datasets ranging in community composition and complexity, was applied successfully to data generated from spiked environmental and clinical samples, and robustly demonstrates superior performance compared with other available tools.
    Keywords: Genomics
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  • 5
    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
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    Topics: Biology
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  • 6
    Publication Date: 2016-07-09
    Description: Chromosome-long haplotyping of human genomes is important to identify genetic variants with differing gene expression, in human evolution studies, clinical diagnosis, and other biological and medical fields. Although several methods have realized haplotyping based on sequencing technologies or population statistics, accuracy and cost are factors that prohibit their wide use. Borrowing ideas from group testing theories, we proposed a clone-based haplotyping method by overlapping pool sequencing. The clones from a single individual were pooled combinatorially and then sequenced. According to the distinct pooling pattern for each clone in the overlapping pool sequencing, alleles for the recovered variants could be assigned to their original clones precisely. Subsequently, the clone sequences could be reconstructed by linking these alleles accordingly and assembling them into haplotypes with high accuracy. To verify the utility of our method, we constructed 130 110 clones in silico for the individual NA12878 and simulated the pooling and sequencing process. Ultimately, 99.9% of variants on chromosome 1 that were covered by clones from both parental chromosomes were recovered correctly, and 112 haplotype contigs were assembled with an N50 length of 3.4 Mb and no switch errors. A comparison with current clone-based haplotyping methods indicated our method was more accurate.
    Keywords: Genomics
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  • 7
    Publication Date: 2016-07-28
    Description: Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these ‘repressed transcripts’ have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.
    Keywords: Genomics
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  • 8
    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
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  • 9
    Publication Date: 2013-06-08
    Description: DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine–guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.
    Keywords: Genomics
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  • 10
    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
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  • 11
    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
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  • 12
    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
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  • 13
    Publication Date: 2014-12-17
    Description: The advent in high-throughput-sequencing (HTS) technologies has revolutionized conventional biodiversity research by enabling parallel capture of DNA sequences possessing species-level diagnosis. However, polymerase chain reaction (PCR)-based implementation is biased by the efficiency of primer binding across lineages of organisms. A PCR-free HTS approach will alleviate this artefact and significantly improve upon the multi-locus method utilizing full mitogenomes. Here we developed a novel multiplex sequencing and assembly pipeline allowing for simultaneous acquisition of full mitogenomes from pooled animals without DNA enrichment or amplification. By concatenating assemblies from three de novo assemblers, we obtained high-quality mitogenomes for all 49 pooled taxa, with 36 species 〉15 kb and the remaining 〉10 kb, including 20 complete mitogenomes and nearly all protein coding genes (99.6%). The assembly quality was carefully validated with Sanger sequences, reference genomes and conservativeness of protein coding genes across taxa. The new method was effective even for closely related taxa, e.g. three Drosophila spp., demonstrating its broad utility for biodiversity research and mito-phylogenomics. Finally, the in silico simulation showed that by recruiting multiple mito-loci, taxon detection was improved at a fixed sequencing depth. Combined, these results demonstrate the plausibility of a multi-locus mito-metagenomics approach as the next phase of the current single-locus metabarcoding method.
    Keywords: Genomics
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  • 14
    Publication Date: 2014-12-17
    Description: Chromatin modifiers and histone modifications are components of a chromatin-signaling network involved in transcription and its regulation. The interactions between chromatin modifiers and histone modifications are often unknown, are based on the analysis of few genes or are studied in vitro . Here, we apply computational methods to recover interactions between chromatin modifiers and histone modifications from genome-wide ChIP-Seq data. These interactions provide a high-confidence backbone of the chromatin-signaling network. Many recovered interactions have literature support; others provide hypotheses about yet unknown interactions. We experimentally verified two of these predicted interactions, leading to a link between H4K20me1 and members of the Polycomb Repressive Complexes 1 and 2. Our results suggest that our computationally derived interactions are likely to lead to novel biological insights required to establish the connectivity of the chromatin-signaling network involved in transcription and its regulation.
    Keywords: Genomics
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  • 15
    Publication Date: 2015-04-02
    Description: With read lengths of currently up to 2 x 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.
    Keywords: Genomics
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  • 16
    Publication Date: 2015-04-02
    Description: RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se , other technologies are needed to more precisely determine acute transcriptional responses. Here, we have developed an easy, sensitive and accurate novel computational method, iRNA-seq , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data. Comparison of the results derived from iRNA-seq analyses with parallel results derived using current methods for genome-wide determination of transcriptional activity, i.e. global run-on (GRO)-seq and RNA polymerase II (RNAPII) ChIP-seq, demonstrate that iRNA-seq provides similar results in terms of number of regulated genes and their fold change. However, unlike the current methods that are all very labor-intensive and demanding in terms of sample material and technologies, iRNA-seq is cheap and easy and requires very little sample material. In conclusion, iRNA-seq offers an attractive novel alternative to current methods for determination of changes in transcriptional activity at a genome-wide level.
    Keywords: Genomics
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  • 17
    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
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  • 18
    Publication Date: 2015-12-29
    Description: This article applies the concept of a term structure to agricultural land rental prices. Based on theoretical considerations, we develop a hedonic pricing model that allows for different shapes of the term structure curve while controlling for other price-relevant characteristics. We apply this model to land lease contracts in Saxony-Anhalt. We find an upward-sloping term structure during the agricultural price boom in 2007 and 2008, where market participants expected increasing rental prices. For the subsequent years, however, we detect a single-humped term structure. Hence, market participants revised their expectations and assumed a decline of land rental prices in the long term.
    Keywords: D44 - Auctions, E43 - Determination of Interest Rates ; Term Structure of Interest Rates, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 19
    Publication Date: 2016-01-09
    Description: Identifying large-scale structural variation in cancer genomes continues to be a challenge to researchers. Current methods rely on genome alignments based on a reference that can be a poor fit to highly variant and complex tumor genomes. To address this challenge we developed a method that uses available breakpoint information to generate models of structural variations. We use these models as references to align previously unmapped and discordant reads from a genome. By using these models to align unmapped reads, we show that our method can help to identify large-scale variations that have been previously missed.
    Keywords: Genomics
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  • 20
    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
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  • 21
    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
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  • 22
    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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  • 23
    Publication Date: 2015-08-29
    Description: Any given human individual carries multiple genetic variants that disrupt protein-coding genes, through structural variation, as well as nucleotide variants and indels. Predicting the phenotypic consequences of a gene disruption remains a significant challenge. Current approaches employ information from a range of biological networks to predict which human genes are haploinsufficient (meaning two copies are required for normal function) or essential (meaning at least one copy is required for viability). Using recently available study gene sets, we show that these approaches are strongly biased towards providing accurate predictions for well-studied genes. By contrast, we derive a haploinsufficiency score from a combination of unbiased large-scale high-throughput datasets, including gene co-expression and genetic variation in over 6000 human exomes. Our approach provides a haploinsufficiency prediction for over twice as many genes currently unassociated with papers listed in Pubmed as three commonly-used approaches, and outperforms these approaches for predicting haploinsufficiency for less-studied genes. We also show that fine-tuning the predictor on a set of well-studied ‘gold standard’ haploinsufficient genes does not improve the prediction for less-studied genes. This new score can readily be used to prioritize gene disruptions resulting from any genetic variant, including copy number variants, indels and single-nucleotide variants.
    Keywords: Genomics
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  • 24
    Publication Date: 2015-07-10
    Description: Analyses of the costs of regulating greenhouse gas emissions from dairy production, which could be used to assess the effectiveness of alternative policy measures, is a missing link in the literature. This article addresses this gap by establishing the economic impact associated with a hypothetical greenhouse gas environmental regulatory regime across major dairy producing counties in the United States. In doing so, the article makes three important contributions to the literature. First, it develops a comprehensive pollution index based on Environmental Protection Agency methodologies, which contrasts with previous studies that rely on partial measures based only on surplus nitrogen stemming from the over-application of fertilizer. Second, the article uses a directional output distance function, an approach that has not been employed previously to evaluate polluting technologies in the U.S. dairy sector. Third, the article incorporates a four-way error approach that accounts for unobserved county heterogeneity, time-invariant persistent technical efficiency, time-varying transient technical efficiency, and a random error. The results indicate that regulating greenhouse gas emissions from dairy farming would induce a 5-percentage point increase in average technical efficiency. In addition, the economic costs of implementing this hypothetical regulatory framework exhibit significant spatial variation across counties in the United States.
    Keywords: D22 - Firm Behavior: Empirical Analysis, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q52 - Pollution Control Costs ; Distributional Effects ; Employment Effects
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 25
    Publication Date: 2015-07-12
    Description: Meta-analysis of gene expression has enabled numerous insights into biological systems, but current methods have several limitations. We developed a method to perform a meta-analysis using the elastic net, a powerful and versatile approach for classification and regression. To demonstrate the utility of our method, we conducted a meta-analysis of lung cancer gene expression based on publicly available data. Using 629 samples from five data sets, we trained a multinomial classifier to distinguish between four lung cancer subtypes. Our meta-analysis-derived classifier included 58 genes and achieved 91% accuracy on leave-one-study-out cross-validation and on three independent data sets. Our method makes meta-analysis of gene expression more systematic and expands the range of questions that a meta-analysis can be used to address. As the amount of publicly available gene expression data continues to grow, our method will be an effective tool to help distill these data into knowledge.
    Keywords: Genomics
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  • 26
    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
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  • 27
    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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  • 28
    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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  • 29
    Publication Date: 2016-08-20
    Description: Advanced sequencing technologies have generated a plethora of data for many chromatin marks in multiple tissues and cell types, yet there is lack of a generalized tool for optimal utility of those data. A major challenge is to quantitatively model the epigenetic dynamics across both the genome and many cell types for understanding their impacts on differential gene regulation and disease. We introduce IDEAS, an i ntegrative and d iscriminative e pigenome a nnotation s ystem, for jointly characterizing epigenetic landscapes in many cell types and detecting differential regulatory regions. A key distinction between our method and existing state-of-the-art algorithms is that IDEAS integrates epigenomes of many cell types simultaneously in a way that preserves the position-dependent and cell type-specific information at fine scales, thereby greatly improving segmentation accuracy and producing comparable annotations across cell types.
    Keywords: Genomics
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  • 30
    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
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  • 31
    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
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  • 32
    Publication Date: 2016-09-03
    Description: A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 x 10 –4 insertions per genome per generation and the rate of IS homologous recombination is 4.5 x 10 –5 recombinations per genome per generation. These events are mostly contributed by the IS elements IS 1 , IS 2 , IS 5 and IS 186 . Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS 186 target-sites, 5'-GGGG(N6/N7)CCCC-3'. We also detected 48 long deletions not involving IS elements.
    Keywords: Genomics
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  • 33
    Publication Date: 2016-07-09
    Description: If agricultural subsidies are largely capitalized into farmland values through their effect on rental rates, then expanding support for agriculture may not benefit farmers who rent the land they farm. Existing evidence on the incidence of subsidies on cash rental rates is mixed. Identification is obscured by unobserved or imprecisely measured factors that tend to be correlated with subsidies, especially land quality and time-varying factors like commodity prices and adverse weather events. A problem that has received less attention is the fact that subsidies and land quality on rented land may differ from owned land. Since most farms possess both rented and owned acreage, farm-level measures of subsidies, land values, and rental rates may bias estimated incidence. Using a new, field-level data set that, for the first time, precisely links subsidies to land parcels, we show that this bias is considerable: where farm-level estimates suggest an incidence of 42–49 cents of the marginal subsidy dollar, field-level estimates from the same farms indicate that landlords capture just 20–28 cents. The size of the farm and the duration of the rental arrangement have substantial effects. Incidence falls by 5–15 cents when doubling total operated acres, and the incidence falls by 0.1–0.8 cents with each additional year of the rental arrangement. Low incidence of subsidies on rents combined with the farm-size and duration effects suggest that farmers renting land have monopsony power.
    Keywords: H22 - Incidence, Q14 - Agricultural Finance, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 34
    Publication Date: 2015-04-21
    Description: Extensive and multi-dimensional data sets generated from recent cancer omics profiling projects have presented new challenges and opportunities for unraveling the complexity of cancer genome landscapes. In particular, distinguishing the unique complement of genes that drive tumorigenesis in each patient from a sea of passenger mutations is necessary for translating the full benefit of cancer genome sequencing into the clinic. We address this need by presenting a data integration framework (OncoIMPACT) to nominate patient-specific driver genes based on their phenotypic impact. Extensive in silico and in vitro validation helped establish OncoIMPACT's robustness, improved precision over competing approaches and verifiable patient and cell line specific predictions (2/2 and 6/7 true positives and negatives, respectively). In particular, we computationally predicted and experimentally validated the gene TRIM24 as a putative novel amplified driver in a melanoma patient. Applying OncoIMPACT to more than 1000 tumor samples, we generated patient-specific driver gene lists in five different cancer types to identify modes of synergistic action. We also provide the first demonstration that computationally derived driver mutation signatures can be overall superior to single gene and gene expression based signatures in enabling patient stratification and prognostication. Source code and executables for OncoIMPACT are freely available from http://sourceforge.net/projects/oncoimpact .
    Keywords: Genomics
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  • 35
    Publication Date: 2015-05-20
    Description: Analysis of rewired upstream subnetworks impacting downstream differential gene expression aids the delineation of evolving molecular mechanisms. Cumulative statistics based on conventional differential correlation are limited for subnetwork rewiring analysis since rewiring is not necessarily equivalent to change in correlation coefficients. Here we present a computational method ChiNet to quantify subnetwork rewiring by statistical heterogeneity that enables detection of potential genotype changes causing altered transcription regulation in evolving organisms. Given a differentially expressed downstream gene set, ChiNet backtracks a rewired upstream subnetwork from a super-network including gene interactions known to occur under various molecular contexts. We benchmarked ChiNet for its high accuracy in distinguishing rewired artificial subnetworks, in silico yeast transcription-metabolic subnetworks, and rewired transcription subnetworks for Candida albicans versus Saccharomyces cerevisiae , against two differential-correlation based subnetwork rewiring approaches. Then, using transcriptome data from tolerant S. cerevisiae strain NRRL Y-50049 and a wild-type intolerant strain, ChiNet identified 44 metabolic pathways affected by rewired transcription subnetworks anchored to major adaptively activated transcription factor genes YAP1 , RPN4 , SFP1 and ROX1 , in response to toxic chemical challenges involved in lignocellulose-to-biofuels conversion. These findings support the use of ChiNet in rewiring analysis of subnetworks where differential interaction patterns resulting from divergent nonlinear dynamics abound.
    Keywords: Genomics
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  • 36
    Publication Date: 2015-05-21
    Description: We review evidence regarding the size and evolution of the "land rush" in the wake of the 2007–8 boom in agricultural commodity prices, and we study the determinants of foreign land acquisition for large-scale agricultural investment. The use of data on bilateral investment relationships to estimate gravity models of transnational land-intensive investments confirms the central role of agro-ecological potential as a pull factor. However, this finding contrasts the standard literature insofar as the quality of the destination country's business climate is insignificant, and weak tenure security is associated with increased interest for investors to acquire land in the country. Policy implications are discussed.
    Keywords: F21 - International Investment ; Long-Term Capital Movements, O13 - Agriculture ; Natural Resources ; Energy ; Environment ; Other Primary Products, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q34 - Natural Resources and Domestic and International Conflicts
    Print ISSN: 0258-6770
    Electronic ISSN: 1564-698X
    Topics: Economics
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  • 37
    Publication Date: 2015-04-18
    Description: Increasing aridity, more frequent and intense drought, and greater degrees of water scarcity create unique challenges for agriculture. In response to these challenges, which often manifest themselves as lower and more variable surface water supplies, as well as depleted and degraded ground water supplies, growers tend to seek opportunities to adapt. One option for growers to reduce their exposure to water scarcity and heightened uncertainty is to diversify. Indeed, access to a portfolio of supplies is one way in which water and irrigation districts, as well as individual growers, are responding to the changing landscape of water resource availability. This article evaluates the benefits to irrigated agriculture from having access to multiple sources of water. With farm-level information on 1,900 agricultural parcels across California, we use the hedonic property value method to investigate the extent that growers benefit from having access to multiple sources of water (i.e., a water portfolio). Our results suggest that while lower quality waters, less reliable water, and less water all negatively impact agricultural land values, holding a water portfolio has a positive impact on land values through its role in mitigating the negative aspects of these factors and reducing the sensitivity of agriculture to climate-related factors. From a policy perspective, such results identify a valuable adaptation tool that irrigation districts may consider to help offset the negative impacts of climate change, drought, and population increases on water supply availability and reliability.
    Keywords: Q10 - General, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q50 - General, Q51 - Valuation of Environmental Effects
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 38
    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
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  • 39
    Publication Date: 2015-06-24
    Description: Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein–RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein–rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a ‘proof of concept’ that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites.
    Keywords: Genomics
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  • 40
    Publication Date: 2015-02-18
    Description: The emergence of new sequencing technologies has facilitated the use of bacterial whole genome alignments for evolutionary studies and outbreak analyses. These datasets, of increasing size, often include examples of multiple different mechanisms of horizontal sequence transfer resulting in substantial alterations to prokaryotic chromosomes. The impact of these processes demands rapid and flexible approaches able to account for recombination when reconstructing isolates’ recent diversification. Gubbins is an iterative algorithm that uses spatial scanning statistics to identify loci containing elevated densities of base substitutions suggestive of horizontal sequence transfer while concurrently constructing a maximum likelihood phylogeny based on the putative point mutations outside these regions of high sequence diversity. Simulations demonstrate the algorithm generates highly accurate reconstructions under realistically parameterized models of bacterial evolution, and achieves convergence in only a few hours on alignments of hundreds of bacterial genome sequences. Gubbins is appropriate for reconstructing the recent evolutionary history of a variety of haploid genotype alignments, as it makes no assumptions about the underlying mechanism of recombination. The software is freely available for download at github.com/sanger-pathogens/Gubbins , implemented in Python and C and supported on Linux and Mac OS X.
    Keywords: Genomics
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  • 41
    Publication Date: 2015-02-18
    Description: Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.
    Keywords: Genomics
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  • 42
    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
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  • 43
    Publication Date: 2012-08-03
    Description: This paper analyses the impact of the recent decision by the European Union to ‘decouple’ agricultural support payments from agricultural production on Irish farmers' land market decisions. The land market participation decisions of Irish farmers are modelled using a dynamic probit model, while the extent of participation decisions is modelled using a dynamic tobit model. Decoupling does not appear to have significantly altered farmers' land market decisions. One likely explanation for this is the cross-compliance obligation for farmers to maintain land in a state fit for agricultural production in order to receive their full payments.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 44
    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
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  • 45
    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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  • 46
    Publication Date: 2016-02-20
    Description: Genetic variants in or near miRNA genes can have profound effects on miRNA expression and targeting. As user-friendly software for the impact prediction of miRNA variants on a large scale is still lacking, we created a tool called miRVaS. miRVaS automates this prediction by annotating the location of the variant relative to functional regions within the miRNA hairpin (seed, mature, loop, hairpin arm, flanks) and by annotating all predicted structural changes within the miRNA due to the variant. In addition, the tool defines the most important region that is predicted to have structural changes and calculates a conservation score that is indicative of the reliability of the structure prediction. The output is presented in a tab-separated file, which enables fast screening, and in an html file, which allows visual comparison between wild-type and variant structures. All separate images are provided for downstream use. Finally, we tested two different approaches on a small test set of published functionally validated genetic variants for their capacity to predict the impact of variants on miRNA expression.
    Keywords: Genomics
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  • 47
    Publication Date: 2016-02-20
    Description: Analysis of RNA-seq data often detects numerous ‘non-co-linear’ (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts ( trans -spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method (‘NCLscan’), which utilized a stepwise alignment strategy to almost completely eliminate false calls (〉98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans -spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans -spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome.
    Keywords: Genomics
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  • 48
    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
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  • 49
    Publication Date: 2015-12-02
    Description: Detecting allelic biases from high-throughput sequencing data requires an approach that maximises sensitivity while minimizing false positives. Here, we present Allelome.PRO, an automated user-friendly bioinformatics pipeline, which uses high-throughput sequencing data from reciprocal crosses of two genetically distinct mouse strains to detect allele-specific expression and chromatin modifications. Allelome.PRO extends approaches used in previous studies that exclusively analyzed imprinted expression to give a complete picture of the ‘allelome’ by automatically categorising the allelic expression of all genes in a given cell type into imprinted, strain-biased, biallelic or non-informative. Allelome.PRO offers increased sensitivity to analyze lowly expressed transcripts, together with a robust false discovery rate empirically calculated from variation in the sequencing data. We used RNA-seq data from mouse embryonic fibroblasts from F1 reciprocal crosses to determine a biologically relevant allelic ratio cutoff, and define for the first time an entire allelome. Furthermore, we show that Allelome.PRO detects differential enrichment of H3K4me3 over promoters from ChIP-seq data validating the RNA-seq results. This approach can be easily extended to analyze histone marks of active enhancers, or transcription factor binding sites and therefore provides a powerful tool to identify candidate cis regulatory elements genome wide.
    Keywords: Genomics
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  • 50
    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
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  • 51
    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
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  • 52
    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
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  • 53
    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
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  • 54
    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
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  • 55
    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
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  • 56
    Publication Date: 2014-01-22
    Description: Water theft carried out by manipulating water meters constrains volumetric pricing in semi-arid regions. Cooperative management can reduce theft and improve incentives for efficient water use by inducing peer monitoring. Using a theoretical model, we show that theft is more likely when prices are high, punishments are weak, and cooperatives are large. We also show how cooperative membership and punishment levels are determined endogenously by constraints on monitoring. We test the model on data from Tunisia for the years 2001–2003, relying on instruments that proxy for unobservable monitoring costs. The results confirm that well-designed incentives can reduce theft, and that constraints on monitoring costs affect institutional design.
    Keywords: D82 - Asymmetric and Private Information, Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q25 - Water
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    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 57
    Publication Date: 2014-01-22
    Description: A large variety of subsidized crop insurance products are available to U.S. crop growers. Distinct and perhaps puzzling patterns in the choices of insurance products and coverage levels can be discerned. Where production conditions are better and yields are less risky then ( a ) higher insurance coverage levels are chosen; and ( b ) revenue insurance is preferred over yield insurance. Also, ( c ) the extent of preference for revenue insurance is stronger in more productive areas. Assuming, as many do, that growers seek to maximize subsidy transfers, point ( a ) can be explained by the interaction between yield technology and natural resource endowments. Points ( b ) and ( c ) can be explained by location in conjunction with the "natural hedge" and a contract design bias in how revenue insurance guarantees are computed. Empirical study of Risk Management Agency data on corn, soybean, and wheat yields, and insurance contract choices lend support to our model inferences.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q24 - Land
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    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 58
    Publication Date: 2013-12-07
    Description: Identity by descent (IBD) can be reliably detected for long shared DNA segments, which are found in related individuals. However, many studies contain cohorts of unrelated individuals that share only short IBD segments. New sequencing technologies facilitate identification of short IBD segments through rare variants, which convey more information on IBD than common variants. Current IBD detection methods, however, are not designed to use rare variants for the detection of short IBD segments. Short IBD segments reveal genetic structures at high resolution. Therefore, they can help to improve imputation and phasing, to increase genotyping accuracy for low-coverage sequencing and to increase the power of association studies. Since short IBD segments are further assumed to be old, they can shed light on the evolutionary history of humans. We propose HapFABIA, a computational method that applies biclustering to identify very short IBD segments characterized by rare variants. HapFABIA is designed to detect short IBD segments in genotype data that were obtained from next-generation sequencing, but can also be applied to DNA microarray data. Especially in next-generation sequencing data, HapFABIA exploits rare variants for IBD detection. HapFABIA significantly outperformed competing algorithms at detecting short IBD segments on artificial and simulated data with rare variants. HapFABIA identified 160 588 different short IBD segments characterized by rare variants with a median length of 23 kb (mean 24 kb) in data for chromosome 1 of the 1000 Genomes Project. These short IBD segments contain 752 000 single nucleotide variants (SNVs), which account for 39% of the rare variants and 23.5% of all variants. The vast majority—152 000 IBD segments—are shared by Africans, while only 19 000 and 11 000 are shared by Europeans and Asians, respectively. IBD segments that match the Denisova or the Neandertal genome are found significantly more often in Asians and Europeans but also, in some cases exclusively, in Africans. The lengths of IBD segments and their sharing between continental populations indicate that many short IBD segments from chromosome 1 existed before humans migrated out of Africa. Thus, rare variants that tag these short IBD segments predate human migration from Africa. The software package HapFABIA is available from Bioconductor. All data sets, result files and programs for data simulation, preprocessing and evaluation are supplied at http://www.bioinf.jku.at/research/short-IBD .
    Keywords: Genomics
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  • 59
    Publication Date: 2013-12-07
    Description: Glioma is the most common and fatal primary brain tumour with poor prognosis; however, the functional roles of miRNAs in glioma malignant progression are insufficiently understood. Here, we used an integrated approach to identify miRNA functional targets during glioma malignant progression by combining the paired expression profiles of miRNAs and mRNAs across 160 Chinese glioma patients, and further constructed the functional miRNA–mRNA regulatory network. As a result, most tumour-suppressive miRNAs in glioma progression were newly discovered, whose functions were widely involved in gliomagenesis. Moreover, three miRNA signatures, with different combinations of hub miRNAs (regulations≥30) were constructed, which could independently predict the survival of patients with all gliomas, high-grade glioma and glioblastoma. Our network-based method increased the ability to identify the prognostic biomarkers, when compared with the traditional method and random conditions. Hsa-miR-524-5p and hsa-miR-628-5p, shared by these three signatures, acted as protective factors and their expression decreased gradually during glioma progression. Functional analysis of these miRNA signatures highlighted their critical roles in cell cycle and cell proliferation in glioblastoma malignant progression, especially hsa-miR-524-5p and hsa-miR-628-5p exhibited dominant regulatory activities. Therefore, network-based biomarkers are expected to be more effective and provide deep insights into the molecular mechanism of glioma malignant progression.
    Keywords: Genomics
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  • 60
    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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  • 61
    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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  • 62
    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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  • 63
    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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  • 64
    Publication Date: 2014-02-11
    Description: A major challenge in cancer genomics is uncovering genes with an active role in tumorigenesis from a potentially large pool of mutated genes across patient samples. Here we focus on the interactions that proteins make with nucleic acids, small molecules, ions and peptides, and show that residues within proteins that are involved in these interactions are more frequently affected by mutations observed in large-scale cancer genomic data than are other residues. We leverage this observation to predict genes that play a functionally important role in cancers by introducing a computational pipeline ( http://canbind.princeton.edu ) for mapping large-scale cancer exome data across patients onto protein structures, and automatically extracting proteins with an enriched number of mutations affecting their nucleic acid, small molecule, ion or peptide binding sites. Using this computational approach, we show that many previously known genes implicated in cancers are enriched in mutations within the binding sites of their encoded proteins. By focusing on functionally relevant portions of proteins—specifically those known to be involved in molecular interactions—our approach is particularly well suited to detect infrequent mutations that may nonetheless be important in cancer, and should aid in expanding our functional understanding of the genomic landscape of cancer.
    Keywords: Genomics
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  • 65
    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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  • 66
    Publication Date: 2014-04-03
    Description: Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea ( Arabidopsis petraea subsp. umbrosa ), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica . The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated ( r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.
    Keywords: Genomics
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  • 67
    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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  • 68
    Publication Date: 2014-04-05
    Description: Perpetual conservation easements permanently remove the option to convert existing habitat to more intensive agricultural production. If existing habitat is at threat of conversion, removing the option to convert will reduce land values. In this article, we estimate the land value discount resulting from perpetual habitat conservation easements by using propensity score matching. We find that on the average eased parcel, land values fall by approximately $86 per acre for every acre of eased habitat. On average, our results suggest that landowners have been adequately compensated and conservation agencies have successfully secured habitat at risk of conversion.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
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  • 69
    Publication Date: 2014-04-05
    Description: This paper investigates the spatial effects that the provision of environmental public goods have on residential location choices in a suburban context. Specifically, a spatial general equilibrium framework is developed to analyze the consequences of adopting an agri-environmental policy promoting the provision of positive farming externalities. We use a static monocentric model of an open city where agricultural bid-rents and agricultural amenities vary endogenously in space, and where the positive externalities associated with agricultural production are valued by households. Consistent with empirical evidence of the potential side effects that conservation policies may have in terms of urbanization patterns and land price changes, we show that under certain conditions implementing an agri-environmental policy may promote additional suburban development. Moreover, we demonstrate that the emergence of disconnected suburban areas may be significantly influenced by the location of land regulated by an agri-environmental policy. Finally, we discuss distributional aspects and show that while introducing an agri-environmental policy has a negative impact on most residential land value, it can have positive effects on farmland and residential land located within the regulated areas, suggesting the non-neutrality of such policies regarding the agents’ assets.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, R13 - General Equilibrium and Welfare Economic Analysis of Regional Economies, R14 - Land Use Patterns, R21 - Housing Demand
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  • 70
    Publication Date: 2014-04-05
    Description: We develop a model to evaluate the profitability of controlling rodent damage by placing barn owl nesting boxes in agricultural areas. The model incorporates the spatial patterns of barn owl predation pressure on rodents, and the impact of this predation pressure on nesting choices and agricultural output. We apply the model to data collected in Israel and find the installation of nesting boxes profitable. While this finding indicates that economic policy instruments to enhance the adoption of this biological control method are redundant, it does support stricter regulations on rodent control using rodenticides.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
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  • 71
    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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  • 72
    Publication Date: 2011-12-27
    Description: We investigate farm size inequality in France using agricultural censuses and farm structure surveys at the NUTS3 level (‘départements’) during the period 1970–2007. Using calculated Gini coefficients, we show that farm size inequality has not systematically increased in France. An econometric analysis of the determinants of farm size inequality reveals that policy measures significantly affected farm size inequality, with most of the measures considered decreasing it. Empirical results suggest that the main contributor was the activity of the SAFER (Société d'Aménagement Foncier et d'Etablissement Rural), a specific feature of the French farm structural policy aimed at regulating rural land management. Besides, this research highlights the great complexity of the dynamics underlying the evolution of farm size distribution.
    Keywords: D30 - General, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 73
    Publication Date: 2011-12-27
    Description: This paper analyses implementation policies of environmental quota trade, with the Flemish nutrient production rights as an example. Implementation policies concern the transaction quantity, quota reduction and prevention of speculation. They are analysed with a static and a dynamic multi-agent quota trade model. The static model with discrete non-auctioned quota trade shows that the obligation for quota sellers to entirely stop their production stimulates structural change. The dynamic model version indicates that a flat rate reduction on traded quota and measures taken to prevent speculation combined with too low penalties for overuse stimulate the total production.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
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  • 74
    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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  • 75
    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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  • 76
    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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  • 77
    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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  • 78
    Publication Date: 2014-11-11
    Description: Efficient water management in agriculture is becoming critical due to increasing environmental constraints and global food and bio-energy demands. Farmers may respond to increased water scarcity along three main adjustment margins: a move towards rain-fed agriculture (super-extensive margin) or towards less water-intensive crops (extensive margin), and a reduction in water intensity for irrigated crops (intensive margin). Using a positive mathematical programming model of regional supply calibrated to economic and agronomic information, we decompose the total effect of reduced water availability on these adjustment margins in Beauce, a productive cereal region that relies on a groundwater resource to meet its irrigation needs. For realistic water scarcity scenarios, 57 per cent of the total response is attributable to super-extensive margin adjustments. The extensive margin represents 28 per cent of the total response, while the intensive margin accounts for 15 per cent. Crop-level analysis reveals more subtle adaptation patterns.
    Keywords: Q11 - Aggregate Supply and Demand Analysis ; Prices, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q25 - Water
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 79
    Publication Date: 2014-09-06
    Description: Water quality regulations in the United States apply almost exclusively to point sources. In impaired watersheds where both point and nonpoint sources contribute to pollution, the U.S. Environmental Protection Agency (EPA) is encouraging the use of point-nonpoint trading to reduce the cost of point sources to meet their permit requirement, and to encourage nonpoint sources to voluntarily contribute more towards meeting overall water quality goals. The EPA guidance encourages trading programs to set a nonpoint source eligibility baseline that extracts some "extra" abatement from nonpoint sources. Research has shown that setting an eligibility baseline that is substantially more stringent than current management could discourage nonpoint source participation and significantly hinder trading. In this paper we examine how choosing the eligibility baseline for agricultural sources affects the efficiency goal of trading (reducing costs to point sources), as well as how it affects the EPA goal of encouraging nonpoint abatement. Using data from the Chesapeake Bay Watershed we find that eligibility baselines set to encourage additional nonpoint source abatement reduce the supply of credits in a market; the more stringent the baseline, the fewer the trades and the smaller the overall abatement from nonpoint sources. A subsidy to farmers for reducing the cost of meeting a baseline encourages greater nonpoint source abatement, but may not benefit the trading market.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q20 - General, Q58 - Government Policy
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 80
    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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  • 81
    Publication Date: 2014-12-13
    Description: As a bioinvasion spreads across a landscape from its point of introduction, damages rise roughly with the square of the distance from the original invasion. It is thus generally beneficial, at the landscape scale, to apply eradication or containment controls early if not immediately upon discovery. However, an individual property owner only has incentives to consider the costs and benefits of control on his/her own property rather than potential landscape-scale damages. Bioinvasions will therefore generally be under-controlled in a landscape of independent owners operating under a laissez-faire system. A mechanism is thus needed to induce early cooperative contributions to control costs from beneficiaries who would, without them, be invaded later. We develop a spatially-explicit, integrated model of invasion spread and human behavior to examine how different degrees of spatial cooperation affect patterns of invasion spread and the total costs and damages imposed. We compare individual laissez-faire, cooperative control by adjacent neighbors, and cooperative control by groups including more distant but nearby neighbors. As expected, private laissez-faire control decisions tend to under-control the invasion relative to socially optimal control under most circumstances. But a reasonably high fraction of first best payoffs can be achieved with only a modest geographical reach of cooperation. We also find that less extensive cooperation is needed to control invasions whose costs and damages otherwise lead to the largest externalities (circumstances with costs that are relatively low compared with damages). This suggests that even small amounts of cooperation to control bioinvasions can provide large social benefits.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
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    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 82
    Publication Date: 2014-12-13
    Description: Farmland conservation policies typically use zoning and differentiated taxes to prevent urban development of farmland, but little is known about the effectiveness of these policies. This study adds to current knowledge by examining the impact of British Columbia's Agricultural Land Reserve (ALR), established in 1973, which severely restricts subdivision and nonagricultural uses for more than 4.7 million hectares of farmland. To determine the extent to which the ALR preserves farmland by reducing or removing the development option, a multilevel hedonic pricing model is used to estimate the impact of land use, geographic, and zoning characteristics on farmland value near the capital city of Victoria on Vancouver Island. Using sales data from 1974 through 2008, the model demonstrates a changing ALR impact over time that varies considerably by improved and unimproved land types. In 2008, landowners paid 19% less for the typical improved farmland parcel within the ALR versus that outside it. This suggests that would-be developers expect permanency in the zoning law, and prefer non-ALR zoned land. However, ALR land that is unimproved has a premium of 55%, suggesting that this land is more valuable for agriculture than for development. Farmland located closer to the city or the commuting highway commands a premium if it has a residence on it, with a residence also explaining why smaller agricultural properties sell at higher prices. However, it appears that zoning by itself is insufficient to protect farmland; other policies likely need to be implemented in conjunction with zoning to protect agricultural land.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q28 - Government Policy, R14 - Land Use Patterns
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 83
    Publication Date: 2014-12-13
    Description: In many parts of the world, natural vegetation has been cleared to allow agricultural production. To ensure a long-term flow of ecosystem services without compromising agricultural activities, restoring the environment requires a balance between public and private benefits and costs. Information about private benefits generated by environmental assets can be utilized to identify conservation opportunities on private lands, evaluate environmental projects, and design effective policy instruments. We use a spatio-temporal hedonic model to estimate the private benefits of native vegetation on rural properties in the state of Victoria, Australia. Specifically, we estimate the marginal value of native vegetation on private land and examine how it varies with the extent of vegetation on a property and across a range of property types and sizes. Private benefits of native vegetation are greater per unit area on small and medium-sized properties and smaller on large production-oriented farms. Native vegetation exhibits diminishing marginal benefits as its proportion of a property increases. The current extent of native vegetation cover is lower than the extent that would maximize the amenity value to many landowners. There is scope for improved targeting of investment in the study region by incorporating private benefits of environmental projects into environmental planning processes. Landowners with high marginal private benefits from revegetation would be more willing to participate in a revegetation program. Targeting these landowners would likely provide higher value for money because such projects could be implemented at lower public cost.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 84
    Publication Date: 2015-03-14
    Description: Methods to interpret personal genome sequences are increasingly required. Here, we report a novel framework (EvoTol) to identify disease-causing genes using patient sequence data from within protein coding-regions. EvoTol quantifies a gene's intolerance to mutation using evolutionary conservation of protein sequences and can incorporate tissue-specific gene expression data. We apply this framework to the analysis of whole-exome sequence data in epilepsy and congenital heart disease, and demonstrate EvoTol's ability to identify known disease-causing genes is unmatched by competing methods. Application of EvoTol to the human interactome revealed networks enriched for genes intolerant to protein sequence variation, informing novel polygenic contributions to human disease.
    Keywords: Genomics
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  • 85
    Publication Date: 2015-03-14
    Description: Detecting in vivo transcription factor (TF) binding is important for understanding gene regulatory circuitries. ChIP-seq is a powerful technique to empirically define TF binding in vivo . However, the multitude of distinct TFs makes genome-wide profiling for them all labor-intensive and costly. Algorithms for in silico prediction of TF binding have been developed, based mostly on histone modification or DNase I hypersensitivity data in conjunction with DNA motif and other genomic features. However, technical limitations of these methods prevent them from being applied broadly, especially in clinical settings. We conducted a comprehensive survey involving multiple cell lines, TFs, and methylation types and found that there are intimate relationships between TF binding and methylation level changes around the binding sites. Exploiting the connection between DNA methylation and TF binding, we proposed a novel supervised learning approach to predict TF–DNA interaction using data from base-resolution whole-genome methylation sequencing experiments. We devised beta-binomial models to characterize methylation data around TF binding sites and the background. Along with other static genomic features, we adopted a random forest framework to predict TF–DNA interaction. After conducting comprehensive tests, we saw that the proposed method accurately predicts TF binding and performs favorably versus competing methods.
    Keywords: Genomics
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  • 86
    Publication Date: 2012-10-10
    Description: Enriching target sequences in sequencing libraries via capture hybridization to bait/probes is an efficient means of leveraging the capabilities of next-generation sequencing for obtaining sequence data from target regions of interest. However, homologous sequences from non-target regions may also be enriched by such methods. Here we investigate the fidelity of capture enrichment for complete mitochondrial DNA (mtDNA) genome sequencing by analyzing sequence data for nuclear copies of mtDNA (NUMTs). Using capture-enriched sequencing data from a mitochondria-free cell line and the parental cell line, and from samples previously sequenced from long-range PCR products, we demonstrate that NUMT alleles are indeed present in capture-enriched sequence data, but at low enough levels to not influence calling the authentic mtDNA genome sequence. However, distinguishing NUMT alleles from true low-level mutations (e.g. heteroplasmy) is more challenging. We develop here a computational method to distinguish NUMT alleles from heteroplasmies, using sequence data from artificial mixtures to optimize the method.
    Keywords: Genomics
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  • 87
    Publication Date: 2012-10-10
    Description: Single nucleotide polymorphisms (SNPs) are increasingly used to tag genetic loci associated with phenotypes such as risk of complex diseases. Technically, this is done genome-wide without prior restriction or knowledge of biological feasibility in scans referred to as genome-wide association studies (GWAS). Depending on the linkage disequilibrium (LD) structure at a particular locus, such tagSNPs may be surrogates for many thousands of other SNPs, and it is difficult to distinguish those that may play a functional role in the phenotype from those simply genetically linked. Because a large proportion of tagSNPs have been identified within non-coding regions of the genome, distinguishing functional from non-functional SNPs has been an even greater challenge. A strategy was recently proposed that prioritizes surrogate SNPs based on non-coding chromatin and epigenomic mapping techniques that have become feasible with the advent of massively parallel sequencing. Here, we introduce an R/Bioconductor software package that enables the identification of candidate functional SNPs by integrating information from tagSNP locations, lists of linked SNPs from the 1000 genomes project and locations of chromatin features which may have functional significance. Availability: FunciSNP is available from Bioconductor (bioconductor.org).
    Keywords: Genomics
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  • 88
    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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  • 89
    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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  • 90
    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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  • 91
    Publication Date: 2014-04-15
    Description: Reconstructing the evolutionary relationships of species is a major goal in biology. Despite the increasing number of completely sequenced genomes, a large number of phylogenetic projects rely on targeted sequencing and analysis of a relatively small sample of marker genes. The selection of these phylogenetic markers should ideally be based on accurate predictions of their combined, rather than individual, potential to accurately resolve the phylogeny of interest. Here we present and validate a new phylogenomics strategy to efficiently select a minimal set of stable markers able to reconstruct the underlying species phylogeny. In contrast to previous approaches, our methodology does not only rely on the ability of individual genes to reconstruct a known phylogeny, but it also explores the combined power of sets of concatenated genes to accurately infer phylogenetic relationships of species not previously analyzed. We applied our approach to two broad sets of cyanobacterial and ascomycetous fungal species, and provide two minimal sets of six and four genes, respectively, necessary to fully resolve the target phylogenies. This approach paves the way for the informed selection of phylogenetic markers in the effort of reconstructing the tree of life.
    Keywords: Genomics
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  • 92
    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
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  • 93
    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
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  • 94
    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
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  • 95
    Publication Date: 2014-09-02
    Description: We examine the effects of energy prices on groundwater extraction using an econometric model of a farmer's irrigation water pumping decision that accounts for both the intensive and extensive margins. Our results show that energy prices have an effect on both types of margins. Increasing energy prices would affect crop selection decisions, crop acreage allocation decisions, and farmers’ demand for water. Our estimated total marginal effect, which sums the effects on the intensive and extensive margins, suggests that a $1 per million btu increase in the energy price would decrease water extraction by an individual farmer by 5.89 acre-feet per year, a decrease of 3.6 percent of the average annual extraction rate. Our estimated elasticity of water extraction with respect to energy price is –0.26.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q40 - General
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    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 96
    Publication Date: 2014-09-02
    Description: Recent increases in farm real estate values in the United States have increased farm equity. By exploiting periods of high and low appreciation that caused various increases in wealth for farmers owning various shares of their farmland, we examine whether U.S. grain farmers expanded their acres harvested or acres owned in response to an increase in their land wealth. We find that land wealth had little effect on farm size. However, for similarly-sized farms, a larger ownership share (10 percentage points) led to an increase in the growth of land owned (2 percentage points). Because older farmers own more of the land that they farm, greater land appreciation slows the rate at which younger farmers acquire land relative to older farmers.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
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  • 97
    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
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  • 98
    Publication Date: 2014-08-02
    Description: Under economic structural changes, some households intensify farming while others reduce it, resulting in significant changes in landholding. This paper studies the link between such changes in landholding and household well-being during a period of rapid economic transformation in Vietnam. Using a rural household panel data set, we find a U-shaped relationship between landholding and well-being: both accumulating crop land and moving out of farming are associated with higher household income and expenditure. Notably, these relationships are greater in communes that are less developed, suggesting that the benefits of structural transformation may diminish at higher levels of development.
    Keywords: I30 - General, I32 - Measurement and Analysis of Poverty, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 99
    Publication Date: 2016-05-20
    Description: Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.
    Keywords: Genomics
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  • 100
    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
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