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      Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

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          Abstract

          We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.

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          Most cited references19

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          Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

          Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.
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            Foundations for engineering biology.

            Drew Endy (2005)
            Engineered biological systems have been used to manipulate information, construct materials, process chemicals, produce energy, provide food, and help maintain or enhance human health and our environment. Unfortunately, our ability to quickly and reliably engineer biological systems that behave as expected remains quite limited. Foundational technologies that make routine the engineering of biology are needed. Vibrant, open research communities and strategic leadership are necessary to ensure that the development and application of biological technologies remains overwhelmingly constructive.
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              Synthetic biology: applications come of age

              Key Points Early synthetic biology designs, namely the genetic toggle switch and repressilator, showed that regulatory components can be characterized and assembled to bring about complex, electronics-inspired behaviours in living systems (for example, memory storage and timekeeping). Through the characterization and assembly of genetic parts and biological building blocks, many more devices have been constructed, including switches, memory elements, oscillators, pulse generators, digital logic gates, filters and communication modules. Advances in the field are now allowing expansion beyond small gene networks to the realm of larger biological programs, which hold promise for a wide range of applications, including biosensing, therapeutics and the production of biofuels, pharmaceuticals and biomaterials. Synthetic biosensing circuits consist of sensitive elements that bind analytes and transducer modules that mobilize cellular responses. Balancing these two modules involves engineering modularity and specificity into the various circuits. Biosensor sensitive elements include environment-responsive promoters (transcriptional), RNA aptamers (translational) and protein receptors (post-translational). Biosensor transducer modules include engineered gene networks (transcriptional), non-coding regulatory RNAs (translational) and protein signal-transduction circuits (post-translational). The contributions of synthetic biology to therapeutics include: engineered networks and organisms for disease-mechanism elucidation, drug-target identification, drug-discovery platforms, therapeutic treatment, therapeutic delivery, and drug production and access. In the microbial production of biofuels and pharmaceuticals, synthetic biology has supplemented traditional genetic and metabolic engineering efforts by aiding the construction of optimized biosynthetic pathways. Optimizing metabolic flux through biosynthetic pathways is traditionally accomplished by driving the expression of pathway enzymes with strong, inducible promoters. New synthetic approaches include the rapid diversification of various pathway components, the rational and model-guided assembly of pathway components, and hybrid solutions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2016
                3 March 2016
                : 11
                : 3
                : e0150182
                Affiliations
                [1 ]Raytheon BBN Technologies, Cambridge, MA, United States of America
                [2 ]iGEM Foundation, Cambridge, MA, United States of America
                [3 ]Synthace, London, United Kingdom
                [4 ]Agilent, Santa Clara, CA, United States of America
                Cardiff University, UNITED KINGDOM
                Author notes

                Competing Interests: The authors of this manuscript have read the journal’s policy and have the following competing interests: The authors received no specific funding for this work. The following authors are employed by for-profit companies: Jacob Beal is employed by Raytheon BBN Technologies; Markus Gershater is employed by Synthace; Jim Hollenhorst is employed by Agilent, and their work on this paper was thus indirectly supported by their salaries. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: JB TH-A KdM ML MG RR. Analyzed the data: JB MG JH TH-A. Wrote the paper: JB TH-A MG KdM ML JH RR.

                ¶ Membership list can be found in the Acknowledgments section.

                Article
                PONE-D-15-50980
                10.1371/journal.pone.0150182
                4777433
                26937966
                f1e697e5-41c3-420d-abab-a9532e043654
                © 2016 Beal et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 November 2015
                : 10 February 2016
                Page count
                Figures: 7, Tables: 3, Pages: 22
                Funding
                Raytheon BBN Technologies, Synthace, and Agilent provided support in the form of salaries for authors JB, MG, and JH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section.
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                Biology and Life Sciences
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