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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.

  • Journal article
    Daza-Martin M, Starowicz K, Jamshad M, Tye S, Ronson GE, MacKay HL, Chauhan AS, Walker AK, Stone HR, Beesley JFJ, Coles JL, Garvin AJ, Stewart GS, McCorvie TJ, Zhang X, Densham RM, Morris JRet al., 2019,

    Isomerization of BRCA1-BARD1 promotes replication fork protection

    , NATURE, Vol: 571, Pages: 521-+, ISSN: 0028-0836
  • Journal article
    Wilkinson M, Drabavicius G, Silanskas A, Gasiunas G, Siksnys V, Wigley Det al., 2019,

    Structure of the DNA-bound spacer capture complex of a Type II CRISPR-Cas system

    , Molecular Cell, Vol: 75, Pages: 90-101.e5, ISSN: 1097-2765

    CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2–6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

  • Journal article
    Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgard MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, Freemont PSet al., 2019,

    Building a global alliance of biofoundries (vol 10, 2040, 2019)

    , Nature Communications, Vol: 10, Pages: 1-2, ISSN: 2041-1723
  • Journal article
    Yates LA, Williams RM, Hailemariam S, Ayala R, Burgers P, Zhang Xet al., 2019,

    Structure of nucleotide-bound Tel1ATM reveals the molecular basis of inhibition and structural rationale for disease mutations

    <jats:sec><jats:title>SUMMARY</jats:title><jats:p>Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases central to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia (A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using cryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our structure reveals molecular details of key residues surrounding the nucleotide binding site and provides a structural and molecular basis for its intrinsically low basal activity. We show that the catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatory domain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an open conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also provides a structural rationale for many mutations found in A-T and cancer.</jats:p></jats:sec>

  • Journal article
    Ziesack M, Gibson T, Oliver JKW, Shumaker AM, Hsu BB, Riglar DT, Giessen TW, DiBenedetto N, Bry L, Way JC, Silver PA, Gerber GKet al., 2019,

    Engineered Interspecies Amino Acid Cross-Feeding Increases Population Evenness in a Synthetic Bacterial Consortium

    , MSYSTEMS, Vol: 4, ISSN: 2379-5077
  • Journal article
    Naydich AD, Nangle SN, Bues JJ, Trivedi D, Nissar N, Inniss MC, Niederhuber MJ, Way JC, Silver PA, Riglar DTet al., 2019,

    Synthetic Gene Circuits Enable Systems-Level Biosensor Trigger Discovery at the Host-Microbe Interface.

    , mSystems, Vol: 4, ISSN: 2379-5077

    Engineering synthetic circuits into intestinal bacteria to sense, record, and respond to in vivo signals is a promising new approach for the diagnosis, treatment, and prevention of disease. However, because the design of disease-responsive circuits is limited by a relatively small pool of known biosensors, there is a need for expanding the capacity of engineered bacteria to sense and respond to the host environment. Here, we apply a robust genetic memory circuit in Escherichia coli to identify new bacterial biosensor triggers responding in the healthy and diseased mammalian gut, which may be used to construct diagnostic or therapeutic circuits. We developed a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably reliable identification of responsive biosensor triggers from pooled libraries. By testing libraries of potential triggers-each consisting of a promoter and ribosome binding site (RBS)-and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensor triggers with increased response in the inflamed gut through comparative screening of one of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensor triggers in the gut and provide a platform for disease-specific screening that is capable of contributing to both the understanding and clinical management of intestinal illness.IMPORTANCE The gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the desig

  • Journal article
    Rajakumar PD, Gower G, Suckling L, Kitney R, McClymont D, Freemont Pet al., 2019,

    Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development

    , Slas Technology, Vol: 24, Pages: 291-297, ISSN: 2472-6303

    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
    Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgard MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, Freemont PSet al., 2019,

    Building a global alliance of biofoundries

    , NATURE COMMUNICATIONS, Vol: 10, Pages: 1-4, ISSN: 2041-1723
  • Journal article
    Danson A, Jovanovic M, Buck M, Zhang Xet al., 2019,

    Mechanisms of s54-dependent transcription initiation and regulation

    , Journal of Molecular Biology, ISSN: 0022-2836

    Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.

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