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  • Conference paper
    Kelwick R, Webb AJ, Wang Y, Allan F, Freemont Pet al., 2019,

    ISEV2019 Abstract Book. PT09.10: Protease biomarker detection using functionalised bioplastic-based biosensors

    , ISEV 2019, Publisher: Co-Action Publishing, ISSN: 2001-3078
  • 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
    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
    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
    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, Pages: 1-28, 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.

  • 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
  • Journal article
    Wilkinson OJ, Martín-González A, Kang H, Northall SJ, Wigley DB, Moreno-Herrero F, Dillingham MSet al., 2019,

    CtIP forms a tetrameric dumbbell-shaped particle which bridges complex DNA end structures for double-strand break repair

    , eLife, Vol: 8, ISSN: 2050-084X

    CtIP is involved in the resection of broken DNA during the S and G2 phases of the cell cycle for repair by recombination. Acting with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with complex structures including forked ssDNA overhangs and nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

  • Conference paper
    Appuswamy R, Lebrigand K, Barbry P, Antonini M, Madderson O, Freemont P, MacDonald J, Heinis Tet al., 2019,

    Oligoarchive: Using DNA in the DBMS storage hierarchy

    The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.

  • Conference paper
    Webb AJ, Landeryou T, Kelwick R, Allan F, Emery A, Jensen K, Templeton M, Freemont PSet al., 2019,


    , 68th Annual Meeting of the American-Society-for-Tropical-Medicine-and-Hygiene (ASTMH), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 182-182, ISSN: 0002-9637

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