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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
    Christophides G, Rona L, Cechetto Carlos B, Souza-Neto Jet al., 2019,

    A comprehensive analysis of malaria transmission in Brazil

    , Pathogens and Global Health, Vol: 113, Pages: 1-13, ISSN: 2047-7724

    Malaria remains a serious public health problem in Brazil despite a significant drop in the number of cases in the past decade. We conduct a comprehensive analysis of malaria transmission in Brazil to highlight the epidemiologically most relevant components that could help tackle the disease. We consider factors impacting on the malaria burden and transmission dynamics including the geographical occurrence of both autochthonous and imported infections, the distribution and abundance of malaria vectors and records of natural mosquito infections with Plasmodium. Our analysis identifies three discrete malaria transmission systems related to the Amazon rainforest, Atlantic rainforest and Brazilian coast, respectively. The Amazonian system accounts for 99% of all malaria cases in the country. It is largely due to autochthonous P. vivax and P. falciparum transmission by mosquitoes of the Nyssorhynchus subgenus, primarily Anopheles darlingi. Whilst P. vivax transmission is widespread, P. falciparum transmission is restricted to hotspot areas mostly in the States of Amazonas and Acre. This system is the major source of P. vivax exportation to the extra-Amazonian regions that are also affected by importation of P. falciparum from Africa. The Atlantic system comprises autochthonous P. vivax transmission typically by the bromeliad-associated mosquitoes An. cruzii and An. bellator of the Kerteszia subgenus. An. cruzii also transmits simian malaria parasites to humans. The third, widespread but geographically fragmented, system is found along the Brazilian coast and comprises P. vivax transmission mainly by An. aquasalis. We conclude that these geographically and biologically distinct malaria transmission systems require specific strategies for effective disease control.

  • Conference paper
    Thaore V, Moore S, Polizzi K, Freemont P, Shah N, Kontoravdi Cet al., 2019,

    Cell-free multi-enzyme system for the industrial production of fine chemicals

    , Chemical Engineering Day UK 2019
  • Journal article
    Aw R, Polizzi KM, 2019,

    Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

    , Biotechnology and Bioengineering, Vol: 116, Pages: 656-666, ISSN: 0006-3592

    Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a 3‐fold increase in recombinant protein yield compared to those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg mL‐1. Therefore, this work not only adds to the growing number of CFPS systems from diverse organisms, but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

  • Journal article
    Celinska E, Borkowska M, Bialas W, Kubiak M, Korpys P, Archacka M, Ledesma-Amaro R, Nicaud J-Met al., 2019,

    Genetic engineering of Ehrlich pathway modulate production of higher alcohols in engineered Yarrowia lipolytica

    , FEMS Yeast Research, Vol: 19, ISSN: 1567-1356

    Microbial cells can produce a vast spectrum of chemical compounds, including those most desired by the global chemical market, as for example higher alcohols, which are promising alternative fuels and chemical feedstock. In the current research, we investigated the effects of the Ehrlich pathway genetic engineering on higher alcohols production in Y. lipolytica, which directly follows our previous findings concerning elucidation of putative molecular identities involved in this pathway. To this end, we constructed two alternative expression cassettes composed of previously identified genes, putatively involved in the Ehrlich pathway in Y. lipolytica, and cloned them under the control of constitutive pTEF promoter, and by this-released them from extensive native regulation. The effects of the pathway engineering were investigated upon provision of different, Ehrlich pathway-inducing amino acids (L-Phe, L-Leu, L-Ile and L-Val). In general, amplification of the Ehrlich pathway in many cases led to increased formation of a respective higher alcohol from its precursor. We observed interesting effects of aminotransferase BAT2 deletion on synthesis of 2-phenylethanol and its acetate ester, significant relationship between L-Val and L-Phe catabolic pathways, and extensive 'cross-induction' of the derivative compounds synthesis by non-direct precursors.

  • Journal article
    McAleer MA, Jakasa I, Hurault G, Sarvari P, McLean WHI, Tanaka RJ, Kezic S, Irvine ADet al., 2019,

    Systemic and stratum corneum biomarkers of severity in infant AD include markers of innate and Th-related immunity and angiogenesis

    , British Journal of Dermatology, Vol: 180, Pages: 586-596, ISSN: 1365-2133

    BACKGROUND: Biomarkers of atopic dermatitis (AD) are largely lacking, especially in infant AD. Those that have been examined to date have focused mostly on serum cytokines with few on non-invasive biomarkers in the skin. OBJECTIVES: We aimed to explore biomarkers obtainable from non-invasive sampling of infant skin. We compared these to plasma biomarkers and structural and functional measures of the skin barrier. METHODS: We recruited 100 infants at first presentation with AD, who were treatment naïve to topical or systemic anti-inflammatory therapies and 20 healthy children. We sampled clinically unaffected skin by tape stripping the stratum corneum (SC). Multiple cytokines and chemokines and natural moisturizing factors (NMF) were measured in the SC and plasma. We recorded disease severity and skin barrier function. RESULTS: 19 SC and 12 plasma biomarkers showed significant difference between healthy and AD skin. Some biomarkers were common to both the SC and plasma, and others were compartment-specific. Identified biomarkers of AD severity included Th2 skewed markers (IL-13, CCL17, CCL22, IL-5), markers of innate activation (IL-18, Il-1α, IL1β, CXCL8), angiogenesis (Flt-1, VEGF) and others (sICAM-1, vCAM-1, IL-16, IL-17A). CONCLUSIONS: We identified clinically relevant biomarkers of AD, including novel markers, easily sampled and typed in infants. These markers may provide objective assessment of disease severity and suggest new therapeutic targets, or response measurement targets for AD. Future studies will be required to determine if these biomarkers, seen in very early AD, can predict disease outcomes or comorbidities.

  • Journal article
    Ces O, Elani Y, 2019,

    Community building in synthetic biology.

    , Experimental biology and medicine (Maywood, N.J.), Vol: 244, Pages: 281-282, ISSN: 0037-9727
  • Journal article
    Suckling L, McFarlane C, Sawyer C, Chambers SP, Kitney RI, McClymont DW, Freemont PSet al., 2019,

    Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

    , Synthetic and Systems Biotechnology, Vol: 4, Pages: 57-66, ISSN: 2405-805X

    High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

  • Journal article
    Jamshidiha M, Pérez-Dorado I, Murray JW, Tate EW, Cota E, Read RJet al., 2019,

    Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a

    , Acta Crystallographica Section D Structural Biology, Vol: 75, Pages: 342-353, ISSN: 2059-7983

    Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27a<jats:sup>Mut</jats:sup>, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by <jats:italic>Phaser</jats:italic> were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in <jats:italic>Phaser</jats:italic>. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in <jats:italic>Phaser</jats:italic>, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more sui

  • Journal article
    Kylilis N, Riangrungroj P, Lai H-E, Salema V, Fernández LÁ, Stan G-B, Freemont PS, Polizzi KMet al., 2019,

    Whole-cell biosensor with tuneable limit of detection enables low-cost agglutination assays for medical diagnostic applications

    , ACS Sensors, Vol: 4, Pages: 370-378, ISSN: 2379-3694

    Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date, the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design can detect 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 using straight-forward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanbodies against 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 field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.

  • Journal article
    Taylor G, Mordaka P, Heap J, 2019,

    Start-stop assembly: a functionally scarless DNA assembly system optimized for metabolic engineering

    , Nucleic Acids Research, Vol: 47, Pages: e17-e17, ISSN: 0305-1048

    DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

  • Conference paper
    Girvan P, Teng X, Brooks NJ, Baldwin GS, Ying Let al., 2019,

    Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications

    , 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
  • Journal article
    Poulton J, Wolde PRT, Ouldridge TE, 2019,

    Non-equilibrium correlations in minimal dynamical models of polymer copying

    , Proceedings of the National Academy of Sciences, Vol: 116, Pages: 1946-1951, ISSN: 0027-8424

    Living systems produce "persistent" copies of information-carrying polymers, in which template and copy sequences remain correlated after physically decoupling. We identify a general measure of the thermodynamic efficiency with which these non-equilibrium states are created, and analyze the accuracy and efficiency of a family of dynamical models that produce persistent copies. For the weakest chemical driving, when polymer growth occurs in equilibrium, both the copy accuracy and, more surprisingly, the efficiency vanish. At higher driving strengths, accuracy and efficiency both increase, with efficiency showing one or more peaks at moderate driving. Correlations generated within the copy sequence, as well as between template and copy, store additional free energy in the copied polymer and limit the single-site accuracy for a given chemical work input. Our results provide insight in the design of natural self-replicating systems and can aid the design of synthetic replicators.

  • Journal article
    McCarty NS, Ledesma-Amaro R, 2019,

    Synthetic biology tools to engineer microbial communities for biotechnology

    , Trends in Biotechnology, Vol: 37, Pages: 181-197, ISSN: 0167-7799

    Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial consortia for diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and the development of new tools to construct synthetic consortia and program their behaviors, will vastly expand the functions that can be performed by communities of interacting microorganisms. Here, we review recent advancements in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss ongoing and emerging efforts to apply consortia for various biotechnological applications, and suggest future applications.

  • Conference paper
    Schumacher J, Waite C, Wang B, 2019,

    Synthetic transcription factors allowtuneable synthetic control of the complex bacterial nor regulon

    , EMBO: Creating is Understanding: Synthetic Biology Masters Complexity
  • Journal article
    Friddin M, Bolognesi G, Salehi-Reyhani A, Ces O, Elani Yet al., 2019,

    Direct manipulation of liquid ordered lipid membrane domains using optical traps

    , Nature Communications Chemistry
  • Journal article
    Habtewold T, Tapanelli S, Ellen KG M, Astrid H, Nikolai W, George K Cet al., 2019,

    Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

    , Malaria Journal, Vol: 18, ISSN: 1475-2875

    BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

  • Journal article
    Tosi T, Hoshiga F, Millership C, Singh R, Eldrid C, Patin D, Mengin-Lecreulx D, Thalassinos K, Freemont P, Grundling Aet al., 2019,

    Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM

    , PLoS Pathogens, Vol: 15, ISSN: 1553-7366

    c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

  • Journal article
    Exley K, Reynolds C, Suckling L, Chee SM, Tsipa A, Freemont P, McClymont D, Kitney Ret al., 2019,

    Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway

    , Journal of Biological Engineering, Vol: 13, ISSN: 1754-1611

    BackgroundThe automation of modular cloning methodologies permits the assembly of many genetic designs. Utilising characterised biological parts aids in the design and redesign of genetic pathways. The characterisation information held on datasheets can be used to determine whether a biological part meets the design requirements. To manage the design of genetic pathways, researchers have turned to modelling-based computer aided design software tools.ResultAn automated workflow has been developed for the design and build of heterologous metabolic pathways. In addition, to demonstrate the powers of electronic datasheets we have developed software which can transfer part information from a datasheet to the Design of Experiment software JMP. To this end we were able to use Design of Experiment software to rationally design and test randomised samples from the design space of a lycopene pathway in E. coli. This pathway was optimised by individually modulating the promoter strength, RBS strength, and gene order targets.ConclusionThe use of standardised and characterised biological parts will empower a design-oriented synthetic biology for the forward engineering of heterologous expression systems. A Design of Experiment approach streamlines the design-build-test cycle to achieve optimised solutions in biodesign. Developed automated workflows provide effective transfer of information between characterised information (in the form of datasheets) and DoE software.

  • Journal article
    Gilbert C, Ellis T, 2019,

    Biological engineered living materials - growing functional materials with genetically-programmable properties

    , ACS Synthetic Biology, Vol: 8, Pages: 1-15, ISSN: 2161-5063

    Natural biological materials exhibit remarkable properties: self-assembly from simple raw materials, precise control of morphology, diverse physical and chemical properties, self-repair and the ability to sense-and-respond to environmental stimuli. Despite having found numerous uses in human industry and society, the utility of natural biological materials is limited. But, could it be possible to genetically program microbes to create entirely new and useful biological materials? At the intersection between microbiology, material science and synthetic biology, the emerging field of biological Engineered Living Materials (ELMs) aims to answer this question. Here we review recent efforts to program cells to produce living materials with novel functional properties, focussing on microbial systems that can be engineered to grow materials and on new genetic circuits for pattern formation that could be used to produce the more complex systems of the future.

  • Conference paper
    Appuswamy R, Brigand KL, Barbry P, Antonini M, Madderson O, Freemont P, McDonald J, Heinis Tet al., 2019,

    OligoArchive: Using DNA in the DBMS storage hierarchy

    , CIDR 2019

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