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Synthetic Biology underpins advances in the bioeconomy

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology. 

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.

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  • JOURNAL ARTICLE
    Kelwick RJR, Ricci L, Chee SM, Bell D, Webb A, Freemont Pet al., 2019,

    Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimise several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimising PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customised our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimise in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ∼50%. In cell-free transcription-translation prototyping reactions, GC-MS quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ±0.39 µM), C104 ΔPhaC_C319A (4.62 ±1.31 µM) and C101 ΔPhaC_C319A (2.65 ±1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ±6.58 µM) and these reactions were also used to characterise a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimising PHAs production.

  • BOOK CHAPTER
    Ouldridge TE, Brittain R, ten Wolde PR, 2018,

    The power of being explicit: demystifying work, heat, and free energy in the physics of computation

    , The Interplay of Thermodynamics and Computation in Both Natural and Artificial Systems
  • JOURNAL ARTICLE
    Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, Nolan T, Crisanti Aet al., 2018,

    A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 1062-+, ISSN: 1087-0156
  • JOURNAL ARTICLE
    Kelly CL, Harris AWK, Steel H, Hancock EJ, Heap JT, Papachristodoulou Aet al., 2018,

    Synthetic negative feedback circuits using engineered small RNAs.

    , Nucleic Acids Res, Vol: 46, Pages: 9875-9889

    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.

  • JOURNAL ARTICLE
    Trantidou T, Dekker L, Polizzi K, Ces O, Elani Yet al., 2018,

    Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

    , INTERFACE FOCUS, Vol: 8, ISSN: 2042-8898
  • JOURNAL ARTICLE
    Kylilis N, Riangrungroj P, Lai H-E, Salema V, Fernandez LA, Stan G-B, Freemont P, Polizzi Ket al., 2018,

    A low-cost biological agglutination assay for medical diagnostic applications

    Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

  • JOURNAL ARTICLE
    Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018,

    Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

    , SCIENTIFIC REPORTS, Vol: 8, ISSN: 2045-2322
  • JOURNAL ARTICLE
    Yunus IS, Wichmann J, Woerdenweber R, Lauersen KJ, Kruse O, Jones PRet al., 2018,

    Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel

    , METABOLIC ENGINEERING, Vol: 49, Pages: 201-211, ISSN: 1096-7176
  • JOURNAL ARTICLE
    Aw R, McKay PF, Shattock RJ, Polizzi KMet al., 2018,

    A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization

    , PROTEIN EXPRESSION AND PURIFICATION, Vol: 149, Pages: 43-50, ISSN: 1046-5928
  • JOURNAL ARTICLE
    Oling D, Lawenius L, Shaw W, Clark S, Kettleborough R, Ellis T, Larsson N, Wigglesworth Met al., 2018,

    Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling

    , ACS SYNTHETIC BIOLOGY, Vol: 7, Pages: 2317-2321, ISSN: 2161-5063
  • JOURNAL ARTICLE
    Yunus IS, Jones PR, 2018,

    Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols

    , METABOLIC ENGINEERING, Vol: 49, Pages: 59-68, ISSN: 1096-7176
  • JOURNAL ARTICLE
    Schaerli Y, Jimenez A, Duarte JM, Mihajlovic L, Renggli J, Isalan M, Sharpe J, Wagner Aet al., 2018,

    Synthetic circuits reveal how mechanisms of gene regulatory networks constrain evolution

    , MOLECULAR SYSTEMS BIOLOGY, Vol: 14, ISSN: 1744-4292
  • JOURNAL ARTICLE
    Aw R, McKay PF, Shattock RJ, Polizzi KMet al., 2018,

    A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.

    , Protein Expr Purif, Vol: 149, Pages: 43-50

    Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

  • JOURNAL ARTICLE
    Rajakumar PD, Gowers G-OF, Suckling L, Foster A, Ellis T, Kitney RI, McClymont DW, Freemont PSet al., 2018,

    Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development.

    , SLAS Technol

    Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

  • JOURNAL ARTICLE
    Ceroni F, Ellis T, 2018,

    The challenges facing synthetic biology in eukaryotes

    , NATURE REVIEWS MOLECULAR CELL BIOLOGY, Vol: 19, Pages: 481-482, ISSN: 1471-0072
  • JOURNAL ARTICLE
    Kylilis N, Tuza ZA, Stan G-B, Polizzi KMet al., 2018,

    Tools for engineering coordinated system behaviour in synthetic microbial consortia

    , NATURE COMMUNICATIONS, Vol: 9, ISSN: 2041-1723
  • JOURNAL ARTICLE
    Liu D, Mannan AA, Han Y, Oyarzún DA, Zhang Fet al., 2018,

    Dynamic metabolic control: towards precision engineering of metabolism.

    , J Ind Microbiol Biotechnol, Vol: 45, Pages: 535-543

    Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the "push-pull-block" strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.

  • JOURNAL ARTICLE
    Brodel AK, Isalan M, Jaramillo A, 2018,

    Engineering of biomolecules by bacteriophage directed evolution

    , CURRENT OPINION IN BIOTECHNOLOGY, Vol: 51, Pages: 32-38, ISSN: 0958-1669
  • JOURNAL ARTICLE
    Pan W, Yuan Y, Ljung L, Goncalves J, Stan G-Bet al., 2018,

    Identification of Nonlinear State-Space Systems From Heterogeneous Datasets

    , IEEE TRANSACTIONS ON CONTROL OF NETWORK SYSTEMS, Vol: 5, Pages: 737-747, ISSN: 2325-5870
  • JOURNAL ARTICLE
    Kogenaru M, Isalan M, 2018,

    Drug-Inducible Control of Lethality Genes: A Low Background Destabilizing Domain Architecture Applied to the Gal4-UAS System in Drosophila

    , ACS SYNTHETIC BIOLOGY, Vol: 7, Pages: 1496-1506, ISSN: 2161-5063

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