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      “Deadman” and “Passcode” microbial kill switches for bacterial containment

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          Abstract

          Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safe-guard systems: the Deadman and Passcode kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI/GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

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          Most cited references35

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          Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.

          Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.
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            Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

            The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.
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              The ribosome binding site calculator.

              The Ribosome Binding Site (RBS) Calculator is a design method for predicting and controlling translation initiation and protein expression in bacteria. The method can predict the rate of translation initiation for every start codon in an mRNA transcript. The method may also optimize a synthetic RBS sequence to achieve a targeted translation initiation rate. Using the RBS Calculator, a protein coding sequence's translation rate may be rationally controlled across a 100,000+ fold range. We begin by providing an overview of the potential biotechnology applications of the RBS Calculator, including the optimization of synthetic metabolic pathways and genetic circuits. We then detail the definitions, methodologies, and algorithms behind the RBS Calculator's thermodynamic model and optimization method. Finally, we outline a protocol for precisely measuring steady-state fluorescent protein expression levels. These methods and protocols provide a clear explanation of the RBS Calculator and its uses. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                101231976
                32624
                Nat Chem Biol
                Nat. Chem. Biol.
                Nature chemical biology
                1552-4450
                1552-4469
                25 October 2015
                07 December 2015
                February 2016
                07 June 2016
                : 12
                : 2
                : 82-86
                Affiliations
                [1 ]Institute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA
                [2 ]Harvard–MIT Program in Health Sciences and Technology, Cambridge, Massachusetts 02139, USA
                [3 ]Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA
                [4 ]Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA
                Author notes
                [* ]Corresponding author. jimjc@ 123456mit.edu phone: 617-324-6607 fax: 617-253-7498
                [†]

                These authors contributed equally to this work

                Article
                HHMIMS732216
                10.1038/nchembio.1979
                4718764
                26641934
                c5d4e36b-a93d-4d6e-8f89-1b71a19db3df

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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                Biochemistry
                Biochemistry

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