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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 articleBeal J, Farny NG, Haddock-Angelli T, et al., 2020,
Robust estimation of bacterial cell count from optical density
, COMMUNICATIONS BIOLOGY, Vol: 3- Author Web Link
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- Citations: 72
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Journal articleFrei T, Cella F, Tedeschi F, et al., 2020,
Characterization and mitigation of gene expression burden in mammalian cells
, Nature Communications, Vol: 11, ISSN: 2041-1723Despite 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.
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Journal articleCrone M, Priestman M, Ciechonska M, et 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: 2041-1723The 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.
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Journal articleXu X, Li X, Liu Y, et al., 2020,
Pyruvate-responsive genetic circuits for dynamic control of central metabolism
, Nature Chemical Biology, Vol: 16, Pages: 1261-1268, ISSN: 1552-4450Dynamic 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.
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Conference paperLankinen 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: 1-25DNA 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.
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Journal articleGraham N, Junghans C, Downes R, et 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: 0163-4453OBJECTIVES: 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.
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Journal articleLv X, Zhang C, Cui S, et 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: 1096-7176Enzyme 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.
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Journal articleGreenig M, Melville A, Huntley D, et al., 2020,
Cross-sectional transcriptional analysis of the ageing murine heart
, Frontiers in Molecular Biosciences, Vol: 7, Pages: 1-14, ISSN: 2296-889XCardiovascular 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.
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Journal articleMeccariello A, Krsticevic F, Colonna R, et 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>
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Journal articleGarcia LDA, Jones PR, 2020,
<i>In silico</i>co-factor balance estimation using constraint-based modelling informs metabolic engineering in<i>Escherichia coli</i>
, PLOS COMPUTATIONAL BIOLOGY, Vol: 16, ISSN: 1553-734X- Author Web Link
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- Citations: 7
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Journal articleJi X-J, Ledesma-Amaro R, 2020,
Microbial lipid biotechnology to produce polyunsaturated fatty acids.
, Trends in Biotechnology, Vol: 38, Pages: 832-834, ISSN: 0167-7799Lipids 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.
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Journal articleSelvaraj P, Wenger EA, Bridenbecker D, et 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- Author Web Link
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- Citations: 11
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Journal articlePerin G, Fletcher T, Sagi-Kiss V, et 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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Journal articleKis Z, Kontoravdi K, Dey AK, et al., 2020,
Rapid development and deployment of high-volumevaccines for pandemic response
, Journal of Advanced Manufacturing and Processing, Vol: 2, Pages: 1-10, ISSN: 2637-403XOvercoming pandemics, such as the current Covid‐19 outbreak, requires the manufacture of several billion doses of vaccines within months. This is an extremely challenging task given the constraints in small‐scale manufacturing for clinical trials, clinical testing timelines involving multiple phases and large‐scale drug substance and drug product manufacturing. To tackle these challenges, regulatory processes are fast‐tracked, and rapid‐response manufacturing platform technologies are used. Here, we evaluate the current progress, challenges ahead and potential solutions for providing vaccines for pandemic response at an unprecedented scale and rate. Emerging rapid‐response vaccine platform technologies, especially RNA platforms, offer a high productivity estimated at over 1 billion doses per year with a small manufacturing footprint and low capital cost facilities. The self‐amplifying RNA (saRNA) drug product cost is estimated at below 1 USD/dose. These manufacturing processes and facilities can be decentralized to facilitate production, distribution, but also raw material supply. The RNA platform technology can be complemented by an a priori Quality by Design analysis aided by computational modeling in order to assure product quality and further speed up the regulatory approval processes when these platforms are used for epidemic or pandemic response in the future.
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Journal articleStorch M, Haines MC, Baldwin GS, 2020,
DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology
, Synthetic Biology, Vol: 5, Pages: ysaa010-ysaa010, ISSN: 2397-7000Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50-$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.
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Journal articleIrmisch P, Ouldridge TE, Seidel R, 2020,
Modelling DNA-strand displacement reactions in the presence of base-pair mismatches
, Journal of the American Chemical Society, Vol: 142, Pages: 11451-11463, ISSN: 0002-7863Toehold-mediated strand displacement is the most abundantly used method to achieve dynamic switching in DNA-based nanotechnology. An ‘invader’ strand binds to the ‘toehold’ overhang of a target strand and replaces a target-bound ’incumbent’ strand. Hereby, complementarity of the invader to the single-stranded toehold provides the energetic bias of the reaction. Despite the widespread use of strand displacement reactions for realizing dynamic DNA nanostructures, variants on the basic motif have not been completely characterized. Here we introduce a simple thermodynamic model, which is capable of quantitatively describing the kinetics of strand displacement reactions in the presence of mismatches, using a minimal set of parameters. Furthermore, our model highlights that base pair fraying and internal loop formation are important mechanisms when involving mismatches in the displacement process. Our model should provide a helpful tool for the rational design of strand-displacement reaction networks.
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Journal articleXu X, Liu Y, Du G, et al., 2020,
Microbial chassis development for natural product biosynthesis
, Trends in Biotechnology, Vol: 38, Pages: 779-796, ISSN: 0167-7799Engineering microbial cells to efficiently synthesize high-value-added natural products has received increasing attention in recent years. In this review, we describe the pipeline to build chassis cells for natural product production. First, we discuss recently developed genome mining strategies for identifying and designing biosynthetic modules and compare the characteristics of different host microbes. Then, we summarize state-of-the-art systems metabolic engineering tools for reconstructing and fine-tuning biosynthetic pathways and transport mechanisms. Finally, we discuss the future prospects of building next-generation chassis cells for the production of natural products. This review provides theoretical guidance for the rational design and construction of microbial strains to produce natural products.
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Journal articleWen Z, Ledesma-Amaro R, Lu M, et al., 2020,
Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial <i>Clostridium</i> community
, BIOTECHNOLOGY AND BIOENGINEERING, Vol: 117, Pages: 2008-2022, ISSN: 0006-3592- Author Web Link
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- Citations: 21
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Journal articlePrabhu AA, Ledesma-Amaro R, Lin CSK, et al., 2020,
Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control
, Biotechnology for Biofuels, Vol: 13, ISSN: 1754-6834BackgroundXylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory.ResultsIn the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD600: 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations.ConclusionThe recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of o
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Journal articleBroedel A, Rodrigues R, Jaramillo A, et al., 2020,
Accelerated evolution of a minimal 63-amino acid dual transcription factor
, Science Advances, Vol: 6, Pages: 1-9, ISSN: 2375-2548Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage λ is one of the smallest known repressors (66 amino acids; a.a.) and activators are typically much larger (e.g. λ cI, 237 a.a.). Indeed, previous efforts to engineer a minimal activator from λ Cro resulted in no activity in vivo, in cells. In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as λ cI, in vivo. To achieve this, we develop Phagemid-Assisted Continuous Evolution: PACEmid. We find that a peptide as small as 63 a.a. functions efficiently as an activator and/or repressor. To our knowledge, this is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.
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Work in the IC-CSynB is supported by a wide range of Research Councils, Learned Societies, Charities and more.
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