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  • Journal article
    de Martín Garrido N, Crone MA, Ramlaul K, Simpson PA, Freemont PS, Aylett CHSet al., 2020,

    Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids

    , Molecular Microbiology, Vol: 113, Pages: 143-152, ISSN: 0950-382X

    Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.

  • Conference paper
    Webb AJ, Kelwick R, Wang Y, Heliot A, Templeton MR, Freemont PSet al., 2019,

    AL-PHA beads: bioplastic-bsaed protease biosensors for global health

    , British Society for Parasitology Autumn Symposium, Belfast, UK
  • Journal article
    Lai H-E, Canavan C, Cameron L, Moore S, Danchenko M, Kuiken T, Sekeyová Z, Freemont PSet al., 2019,

    Synthetic biology and the United Nations

    , Trends in Biotechnology, Vol: 37, Pages: 1146-1151, ISSN: 0167-7799

    Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).

  • Journal article
    Ye F, Kotta-Loizou I, Jovanovic M, Liu X, Dryden D, Buck M, Zhang Xet al., 2019,

    Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

    , eLife, Vol: 9, ISSN: 2050-084X

    Abstract Bacteriophage T7 infects Escherichia coli and evades the host defence system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics. Impact statement DNA mimicry Ocr protein, a well-studied T7 phage protein that inhibits host restriction enzymes, can also inhibit host transcription through competing with sigma factors in binding to RNA polymerase.

  • Journal article
    Freemont P, 2019,

    Synthetic biology industry - Data-driven design is creating new opportunities in biotechnology.

    , Emerging Topics in Life Sciences, Vol: 3, Pages: 651-657, ISSN: 2397-8554

    Synthetic biology is a rapidly emerging interdisciplinary research field that is primarily built upon foundational advances in molecular biology combined with engineering design. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies. This has spurned a rapid growth in start-up companies and the new synthetic biology industry is growing rapidly, with start-up companies receiving ~$6.1B investment since 2015 and a global synthetic biology market value estimated to be $14B by 2026. Many of the new start-upscan be grouped within a multi-layer ‘technology stack’. The ‘stack’ comprises a number of technology layers which together can be applied to a diversity of new biotechnology applications like consumer biotechnology products and living therapies. The ‘stack’ also enables new commercial opportunities and value chains similar to the software design and manufacturing revolution of the 20th century. However, synthetic biology industry is at a crucial point, as it now requires recognisable commercial successes in order for the industry to expand and scale, in terms of investment and companies. However, such expansion may directly challenge the ethos of synthetic biology, in terms of open technology sharing and democratisation, which could by accident lead to multi-national corporations and technology monopolies similar to the existing biotechnology/biopharma industry.

  • Journal article
    Riglar DT, Richmond DL, Potvin-Trottier L, Verdegaal AA, Naydich AD, Bakshi S, Leoncini E, Lyon LG, Paulsson J, Silver PAet al., 2019,

    Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator

  • Journal article
    Wood TE, Howard SA, Forster A, Nolan LM, Manoli E, Bullen NP, Yau HCL, Hachani A, Hayward RD, Whitney JC, Vollmer W, Freemont PS, Filloux Aet al., 2019,

    The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology

    , Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247

    The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

  • Journal article
    Ebright RH, Werner F, Zhang X, 2019,

    RNA Polymerase Reaches 60: Transcription Initiation, Elongation, Termination, and Regulation in Prokaryotes

    , JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3945-3946, ISSN: 0022-2836
  • Journal article
    Danson AE, Jovanovic M, Buck M, Zhang Xet al., 2019,

    Mechanisms of σ<SUP>54</SUP>-Dependent Transcription Initiation and Regulation

    , JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3960-3974, ISSN: 0022-2836
  • 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.

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