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      Synthetic negative feedback circuits using engineered small RNAs


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          Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input–output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

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          Regulatory RNAs in bacteria.

          Bacteria possess numerous and diverse means of gene regulation using RNA molecules, including mRNA leaders that affect expression in cis, small RNAs that bind to proteins or base pair with target RNAs, and CRISPR RNAs that inhibit the uptake of foreign DNA. Although examples of RNA regulators have been known for decades in bacteria, we are only now coming to a full appreciation of their importance and prevalence. Here, we review the known mechanisms and roles of regulatory RNAs, highlight emerging themes, and discuss remaining questions.
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            Refinement and standardization of synthetic biological parts and devices.

            The ability to quickly and reliably engineer many-component systems from libraries of standard interchangeable parts is one hallmark of modern technologies. Whether the apparent complexity of living systems will permit biological engineers to develop similar capabilities is a pressing research question. We propose to adapt existing frameworks for describing engineered devices to biological objects in order to (i) direct the refinement and use of biological 'parts' and 'devices', (ii) support research on enabling reliable composition of standard biological parts and (iii) facilitate the development of abstraction hierarchies that simplify biological engineering. We use the resulting framework to describe one engineered biological device, a genetically encoded cell-cell communication receiver named BBa_F2620. The description of the receiver is summarized via a 'datasheet' similar to those widely used in engineering. The process of refinement and characterization leading to the BBa_F2620 datasheet may serve as a starting template for producing many standardized genetically encoded objects.
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              Synthetic biology: new engineering rules for an emerging discipline

              Synthetic biologists engineer complex artificial biological systems to investigate natural biological phenomena and for a variety of applications. We outline the basic features of synthetic biology as a new engineering discipline, covering examples from the latest literature and reflecting on the features that make it unique among all other existing engineering fields. We discuss methods for designing and constructing engineered cells with novel functions in a framework of an abstract hierarchy of biological devices, modules, cells, and multicellular systems. The classical engineering strategies of standardization, decoupling, and abstraction will have to be extended to take into account the inherent characteristics of biological devices and modules. To achieve predictability and reliability, strategies for engineering biology must include the notion of cellular context in the functional definition of devices and modules, use rational redesign and directed evolution for system optimization, and focus on accomplishing tasks using cell populations rather than individual cells. The discussion brings to light issues at the heart of designing complex living systems and provides a trajectory for future development.

                Author and article information

                Nucleic Acids Res
                Nucleic Acids Res
                Nucleic Acids Research
                Oxford University Press
                12 October 2018
                13 September 2018
                13 September 2018
                : 46
                : 18
                : 9875-9889
                [1 ]Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
                [2 ]Imperial College Centre for Synthetic Biology, Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
                Author notes
                To whom correspondence should be addressed. Tel: +44 1865283036; Email: antonis@ 123456eng.ox.ac.uk . Correspondence may also be addressed to Ciarán L. Kelly. Email: ciaranlk@ 123456gmail.com

                The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.

                Present addresses:

                Ciarán L. Kelly, School of Natural and Environmental Sciences, Newcastle University, Newcastle NE1 7RU, UK.

                Edward J. Hancock, School of Mathematics and Statistics, The University of Sydney, Sydney, NSW 2006, Australia and Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006, Australia.

                Author information
                © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                : 06 September 2018
                : 01 September 2018
                : 05 September 2017
                Page count
                Pages: 15
                Funded by: Engineering and Physical Sciences Research Council 10.13039/501100000266
                Award ID: EP/M002454/1
                Funded by: Biotechnology and Biological Sciences Research Council 10.13039/501100000268
                Award ID: BB/M011321/1
                Synthetic Biology and Bioengineering



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