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      Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates

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          Abstract

          Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply “part mining” to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >10 54 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

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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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            FACS-optimized mutants of the green fluorescent protein (GFP).

            B Cormack, R. Valdivia, S Falkow (1996)
            We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
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              A synthetic biology framework for programming eukaryotic transcription functions.

              Ahmad S. Khalil, Timothy K. Lu, Caleb J. Bashor … (2012)
              Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101231976
                Journal ID (pubmed-jr-id): 32624
                Journal ID (nlm-ta): Nat Chem Biol
                Journal ID (iso-abbrev): Nat. Chem. Biol.
                Title: Nature chemical biology
                ISSN (Print): 1552-4450
                ISSN (Electronic): 1552-4469
                Publication date Nihms-submitted: 11 September 2014
                Publication date (Electronic): 08 December 2013
                Publication date (Print): February 2014
                Publication date PMC-release: 16 September 2014
                Volume: 10
                Issue: 2
                Pages: 99-105
                Affiliations
                [1 ] Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
                [2 ] 1600 4 th Street, San Francisco, CA, 94158, USA
                [3 ] Synthetic Biology R&D Unit, Life Technologies, Carlsbad, CA 92008, USA
                Author notes
                Correspondence and requests for materials should be addressed to C.A.V. ( cavoigt@ 123456gmail.com ).
                Article
                Manuscript ID: NIHMS536568
                DOI: 10.1038/nchembio.1411
                PMC ID: 4165527
                PubMed ID: 24316737
                SO-VID: 76dd2506-aaba-438a-8dd7-04dce5c84402
                License:

                Users may view, print, copy, download and 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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                ScienceOpen disciplines: Biochemistry
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                ScienceOpen disciplines: Biochemistry

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