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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
    Antonakoudis A, Barbosa R, Kotidis P, Kontoravdi Ket al., 2020,

    The era of big data: Genome-scale modelling meets machine learning

    , Computational and Structural Biotechnology Journal, Vol: 18, Pages: 3287-3300, ISSN: 2001-0370

    With omics data being generated at an unprecedented rate, genome-scale modelling has become pivotal in its organisation and analysis. However, machine learning methods have been gaining ground in cases where knowledge is insufficient to represent the mechanisms underlying such data or as a means for data curation prior to attempting mechanistic modelling. We discuss the latest advances in genome-scale modelling and the development of optimisation algorithms for network and error reduction, intracellular constraining and applications to strain design. We further review applications of supervised and unsupervised machine learning methods to omics datasets from microbial and mammalian cell systems and present efforts to harness the potential of both modelling approaches through hybrid modelling.

  • Journal article
    Murray JW, Rutherford AW, Nixon PJ, 2020,

    Photosystem II in a state of disassembly

    , Joule, Vol: 4, Pages: 2082-2084, ISSN: 2542-4351

    The light-driven oxidation of water to oxygen characteristic of oxygenic photosynthesis is catalyzed by a redox-active manganese/calcium cluster embedded in the Photosystem II (PSII) complex. How the cluster is assembled during the biogenesis and repair of PSII is unclear. Cryo-electron microscopy data have now provided new insights into the structure of a PSII complex lacking the cluster and have identified features that might be important for delivery and stabilization of Mn during assembly.

  • Journal article
    Meng F, Ellis T, 2020,

    The second decade of synthetic biology: 2010-2020.

    , Nature Communications, Vol: 11, Pages: 5174-5174, ISSN: 2041-1723

    Synthetic biology is among the most hyped research topics this century, and in2010 it entered its teenage years. But rather than these being a problematictime, we’ve seen synthetic biology blossom and deliver many new technologiesand landmark achievements.

  • Journal article
    Wang J, Ledesma-Amaro R, Wei Y, Ji B, Ji X-Jet al., 2020,

    Metabolic engineering for increased lipid accumulation in Yarrowia lipolytica -A Review

    , Bioresource Technology, Vol: 313, Pages: 1-11, ISSN: 0960-8524

    Current energy security and climate change policies encourage the development and utilization of bioenergy. Oleaginous yeasts provide a particularly attractive platform for the sustainable production of biofuels and industrial chemicals due to their ability to accumulate high amounts of lipids. In particular, microbial lipids in the form of triacylglycerides (TAGs) produced from renewable feedstocks have attracted considerable attention because they can be directly used in the production of biodiesel and oleochemicals analogous to petrochemicals. As an oleaginous yeast that is generally regarded as safe, Yarrowia lipolytica has been extensively studied, with large amounts of data on its lipid metabolism, genetic tools, and genome sequencing and annotation. In this review, we highlight the newest strategies for increasing lipid accumulation using metabolic engineering and summarize the research advances on the overaccumulation of lipids in Y. lipolytica. Finally, perspectives for future engineering approaches are proposed.

  • Conference paper
    Pan K, Hurault G, Arulkumaran K, Williams H, Tanaka Ret al., 2020,

    EczemaNet: automating detection and severity assessment of atopic dermatitis

    , International Workshop on Machine Learning in Medical Imaging, Publisher: Springer Verlag, Pages: 220-230, ISSN: 0302-9743

    Atopic dermatitis (AD), also known as eczema, is one of themost common chronic skin diseases. AD severity is primarily evaluatedbased on visual inspections by clinicians, but is subjective and has largeinter- and intra-observer variability in many clinical study settings. Toaid the standardisation and automating the evaluation of AD severity,this paper introduces a CNN computer vision pipeline, EczemaNet, thatfirst detects areas of AD from photographs and then makes probabilisticpredictions on the severity of the disease. EczemaNet combines trans-fer and multitask learning, ordinal classification, and ensembling overcrops to make its final predictions. We test EczemaNet using a set of im-ages acquired in a published clinical trial, and demonstrate low RMSEwith well-calibrated prediction intervals. We show the effectiveness of us-ing CNNs for non-neoplastic dermatological diseases with a medium-sizedataset, and their potential for more efficiently and objectively evaluatingAD severity, which has greater clinical relevance than mere classification.

  • Journal article
    Price TAR, Windbichler N, Unckless RL, Sutter A, Runge J-N, Ross PA, Pomiankowski A, Nuckolls NL, Montchamp-Moreau C, Mideo N, Martin OY, Manser A, Legros M, Larracuente AM, Holman L, Godwin J, Gemmell N, Courret C, Buchman A, Barrett LG, Lindholm AKet al., 2020,

    Resistance to natural and synthetic gene drive systems

    , JOURNAL OF EVOLUTIONARY BIOLOGY, Vol: 33, Pages: 1345-1360, ISSN: 1010-061X
  • Journal article
    Beal J, Farny NG, Haddock-Angelli T, Selvarajah V, Baldwin GS, Buckley-Taylor R, Gershater M, Kiga D, Marken J, Sanchania V, Sison A, Workman CTet al., 2020,

    Robust estimation of bacterial cell count from optical density

    , COMMUNICATIONS BIOLOGY, Vol: 3
  • Journal article
    Crone MA, Priestman M, Ciechonska M, Jensen K, Sharp DJ, Anand A, Randell P, Storch M, Freemont PSet al., 2020,

    A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics (vol 11, 4464, 2020)

    , NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
  • Journal article
    Frei T, Cella F, Tedeschi F, Gutiérrez J, Stan G-B, Khammash M, Siciliano Vet al., 2020,

    Characterization and mitigation of gene expression burden in mammalian cells

    , Nature Communications, Vol: 11, ISSN: 2041-1723

    Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

  • Journal article
    Crone M, Priestman M, Ciechonska M, Jensen K, Sharp D, Anand A, Randell P, Storch M, Freemont Pet al., 2020,

    A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

    , Nature Communications, Vol: 11, Pages: 1-11, ISSN: 2041-1723

    The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.

  • Journal article
    Xu X, Li X, Liu Y, Zhu Y, Li J, Du G, Chen J, Ledesma-Amaro R, Liu Let al., 2020,

    Pyruvate-responsive genetic circuits for dynamic control of central metabolism

    , Nature Chemical Biology, Vol: 16, Pages: 1261-1268, ISSN: 1552-4450

    Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l−1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l−1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.

  • Conference paper
    Lankinen A, Ruiz IM, Ouldridge TE, 2020,

    Implementing non-equilibrium networks with active circuits of duplex catalysts

    , 26th International Conference on DNA Computing and Molecular Programming (DNA 26), Publisher: Schloss Dagstuhl--Leibniz-Zentrum, Pages: 1-25

    DNA strand displacement (DSD) reactions have been used to construct chemicalreaction networks in which species act catalytically at the level of theoverall stoichiometry of reactions. These effective catalytic reactions aretypically realised through one or more of the following: many-stranded gatecomplexes to coordinate the catalysis, indirect interaction between thecatalyst and its substrate, and the recovery of a distinct ``catalyst'' strandfrom the one that triggered the reaction. These facts make emulation of theout-of-equilibrium catalytic circuitry of living cells more difficult. Here, wepropose a new framework for constructing catalytic DSD networks: ActiveCircuits of Duplex Catalysts (ACDC). ACDC components are all double-strandedcomplexes, with reactions occurring through 4-way strand exchange. Catalystsdirectly bind to their substrates, and and the ``identity'' strand of thecatalyst recovered at the end of a reaction is the same molecule as the onethat initiated it. We analyse the capability of the framework to implementcatalytic circuits analogous to phosphorylation networks in living cells. Wealso propose two methods of systematically introducing mismatches within DNAstrands to avoid leak reactions and introduce driving through net base pairformation. We then combine these results into a compiler to automate theprocess of designing DNA strands that realise any catalytic network allowed byour framework.

  • Journal article
    Graham N, Junghans C, Downes R, Sendall C, Lai H, McKirdy A, Elliott P, Howard R, Wingfield D, Priestman M, Ciechonska M, Cameron L, Storch M, Crone MA, Freemont PS, Randell P, McLaren R, Lang N, Ladhani S, Sanderson F, Sharp DJet al., 2020,

    SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes

    , Journal of Infection, Vol: 81, Pages: 411-419, ISSN: 0163-4453

    OBJECTIVES: To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents. METHODS: An outbreak investigation involving 394 residents and 70 staff, was carried out in 4 nursing homes affected by COVID-19 outbreaks in central London. Two point-prevalence surveys were performed one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing. RESULTS: Overall, 26% (95% CI 22 to 31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70 to 336) compared with previous years. Systematic testing identified 40% (95% CI 35 to 46) of residents as positive for SARS-CoV-2, and of these 43% (95% CI 34 to 52) were asymptomatic and 18% (95% CI 11 to 24) had only atypical symptoms; 4% (95% CI -1 to 9) of asymptomatic staff also tested positive. CONCLUSIONS: The SARS-CoV-2 outbreak in four UK nursing homes was associated with very high infection and mortality rates. Many residents developed either atypical or no discernible symptoms. A number of asymptomatic staff members also tested positive, suggesting a role for regular screening of both residents and staff in mitigating future outbreaks.

  • Journal article
    Lv X, Zhang C, Cui S, Xu X, Wang L, Li J, Du G, Chen J, Ledesma-Amaro R, Liu Let al., 2020,

    Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis

    , Metabolic Engineering, Vol: 61, Pages: 96-105, ISSN: 1096-7176

    Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

  • Journal article
    Greenig M, Melville A, Huntley D, Isalan M, Mielcarek Met al., 2020,

    Cross-sectional transcriptional analysis of the ageing murine heart

    , Frontiers in Molecular Biosciences, Vol: 7, Pages: 1-14, ISSN: 2296-889X

    Cardiovascular disease accounts for millions of deaths each year and is currently the leading cause of mortality worldwide. The ageing process is clearly linked to cardiovascular disease, however, the exact relationship between ageing and heart function is not fully understood. Furthermore, a holistic view of cardiac ageing, linking features of early life development to changes observed in old age, has not been synthesized. Here, we re-purpose RNA-sequencing data previously-collected by our group, investigating gene expression differences between wild-type mice of different age groups that represent key developmental milestones in the murine lifespan. DESeq2’s generalized linear model was applied with two hypothesis6testing approaches to identify differentially-expressed (DE) genes, both between pairs of age groups and across mice of all ages. Pairwise comparisons identified genes associated with specific age transitions, while comparisons across all age groups identified a large set of genes associated with the ageing process more broadly. An unsupervised machine learning approach was then applied to extract common expression patterns from this set of age-associated genes. Sets of genes with both linear and non-linear expression trajectories were identified, suggesting that ageing not only involves the activation of gene expression programs unique to different age groups, but also the re-activation of gene expression programs from earlier ages. Overall, we present a comprehensive transcriptomic analysis of cardiac gene expression patterns across the entirety of the murine lifespan.

  • Journal article
    Meccariello A, Krsticevic F, Colonna R, Del Corsano G, Fasulo B, Papathanos PA, Windbichler Net al., 2020,

    Engineered sex distortion in the global agricultural pest <i>Ceratitis capitata</i>

    <jats:title>Abstract</jats:title><jats:p>Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly <jats:italic>Ceratitis capitata</jats:italic>, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome specific sequence repeats. When targeted by gRNAs in conjunction with Cas9 they yielded a significant and consistent distortion of the sex ratio in independent transgenic strains and a combination of distorters induced a strong bias towards males (~80%). Our results demonstrate the design of sex distorters in a non-model organism and suggest that strains with characteristics suitable for field application could be developed for a range of medically or agriculturally relevant insect species.</jats:p>

  • Journal article
    Garcia LDA, Jones PR, 2020,

    In silicoco-factor balance estimation using constraint-based modelling informs metabolic engineering inEscherichia coli

    , PLOS COMPUTATIONAL BIOLOGY, Vol: 16, ISSN: 1553-734X
  • Journal article
    Selvaraj P, Wenger EA, Bridenbecker D, Windbichler N, Russell JR, Gerardin J, Bever CA, Nikolov Met al., 2020,

    Vector genetics, insecticide resistance and gene drives: An agent-based modeling approach to evaluate malaria transmission and elimination

    , PLOS COMPUTATIONAL BIOLOGY, Vol: 16, ISSN: 1553-734X
  • Journal article
    Ji X-J, Ledesma-Amaro R, 2020,

    Microbial lipid biotechnology to produce polyunsaturated fatty acids.

    , Trends in Biotechnology, Vol: 38, Pages: 832-834, ISSN: 0167-7799

    Lipids rich in polyunsaturated fatty acids are important nutrients. They are traditionally extracted from animals and plants but alternatively can be obtained from microbes through microbial lipid biotechnology. To make this process more economical, apart from strain engineering, the next frontier is through bioprocess and downstream innovation.

  • Journal article
    Perin G, Fletcher T, Sagi-Kiss V, Gaboriau DCA, Carey MR, Bundy JG, Jones PRet al., 2020,

    Calm on the surface, dynamic on the inside. Molecular homeostasis in response to regulatory and metabolic perturbation of<i>Anabaena</i>sp. PCC 7120 nitrogen metabolism

    <jats:title>Abstract</jats:title><jats:p>Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. Δ<jats:italic>amt</jats:italic>mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations.

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