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Synthetic Gene Circuits: Design with Directed Evolution

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      Abstract

      Synthetic circuits offer great promise for generating insights into nature's underlying design principles or forward engineering novel biotechnology applications. However, construction of these circuits is not straightforward. Synthetic circuits generally consist of components optimized to function in their natural context, not in the context of the synthetic circuit. Combining mathematical modeling with directed evolution offers one promising means for addressing this problem. Modeling identifies mutational targets and limits the evolutionary search space for directed evolution, which alters circuit performance without the need for detailed biophysical information. This review examines strategies for integrating modeling and directed evolution and discusses the utility and limitations of available methods.

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      Most cited references 58

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      A general method for numerically simulating the stochastic time evolution of coupled chemical reactions

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        Construction of a genetic toggle switch in Escherichia coli.

        It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements. These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator. However, these behaviours have not been demonstrated in networks of non-specialized regulatory components. Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability. The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network. It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold. As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy.
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          A synthetic oscillatory network of transcriptional regulators.

          Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function. We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks.
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            Author and article information

            Affiliations
            [1 ]Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, California 91125; email:
            Journal
            Annual Review of Biophysics and Biomolecular Structure
            Annu. Rev. Biophys. Biomol. Struct.
            Annual Reviews
            1056-8700
            1545-4266
            June 2007
            June 2007
            : 36
            : 1
            : 1-19
            10.1146/annurev.biophys.36.040306.132600
            © 2007

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