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      Design, construction and characterization of a set of insulated bacterial promoters

      research-article
      Author(s): , , *
      Publication date (Electronic):
      Journal: Nucleic Acids Research
      Publisher: Oxford University Press

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          Abstract

          We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5′- or 3′-sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community.

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

          Dae-Kyun Ro, Eric M Paradise, Mario Ouellet … (2006)
          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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              A synthetic multicellular system for programmed pattern formation.

              Subhayu Basu, Yoram Gerchman, Cynthia H Collins … (2005)
              Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell-cell communication and intracellular signal processing. Here we show a synthetic multicellular system in which genetically engineered 'receiver' cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by 'sender' cells. In receiver cells, 'band-detect' gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated 'lawn' of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Nucleic Acids Res
                Journal ID (publisher-id): nar
                Journal ID (hwp): nar
                Title: Nucleic Acids Research
                Publisher: Oxford University Press
                ISSN (Print): 0305-1048
                ISSN (Electronic): 1362-4962
                Publication date Collection: February 2011
                Publication date (Print): February 2011
                Publication date (Electronic): 15 September 2010
                Publication date PMC-release: 15 September 2010
                Volume: 39
                Issue: 3
                Pages: 1131-1141
                Affiliations
                Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
                Author notes
                *To whom correspondence should be addressed. Tel: +1 617 253 3163; Fax: +1 617 258 0673; Email: bobsauer@ 123456mit.edu
                Article
                Publisher ID: gkq810
                DOI: 10.1093/nar/gkq810
                PMC ID: 3035448
                PubMed ID: 20843779
                SO-VID: 4996bab2-3bfa-45d6-96e0-e0f76a925297
                Copyright © © The Author(s) 2010. Published by Oxford University Press.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 11 May 2010
                Date revision received : 27 August 2010
                Date accepted : 1 September 2010
                Categories
                Subject: Synthetic Biology and Chemistry

                ScienceOpen disciplines: Genetics
                Data availability:
                ScienceOpen disciplines: Genetics

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