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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
    Moya-Ramirez I, Kontoravdi K, Polizzi K, 2019,

    Low-cost and user-friendly biosensor to test the integrity of mRNA molecules suitable for field applications

    , Biosensors and Bioelectronics, Vol: 137, Pages: 199-206, ISSN: 0956-5663

    The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5’ end of the RNA under study and β-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-βLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products.

  • Journal article
    Walker K, Goosens V, Das A, Graham A, Ellis Tet al., 2019,

    Engineered cell-to-cell signalling within growing bacterial cellulose pellicles

    , Microbial Biotechnology, Vol: 12, Pages: 611-619, ISSN: 1751-7915

    Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in health care, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell‐to‐cell communication into a bacterial cellulose‐producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl‐homoserine lactone. We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell‐to‐cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.

  • Journal article
    Kotidis P, Jedrzejewski P, Sou SN, Sellick C, Polizzi K, Del Val IJ, Kontoravdi Cet al., 2019,

    Model-based optimization of antibody galactosylation in CHO cell culture

    , Biotechnology and Bioengineering, Vol: 116, Pages: 1612-1626, ISSN: 0006-3592

    Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding - specifically that of galactose and uridine - on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher-performing bioprocesses.

  • Journal article
    Kis Z, Shattock R, Shah N, Kontoravdi Cet al., 2019,

    Correction: Emerging technologies for low‐cost, rapid vaccine manufacture

    , Biotechnology Journal, Vol: 14, Pages: 1-2, ISSN: 1860-6768
  • Journal article
    Kis Z, Papathanasiou M, CalvoSerrano R, Kontoravdi C, Shah Net al., 2019,

    A model‐based quantification of the impact of new manufacturing technologies on developing country vaccine supply chain performance: A Kenyan case study

    , Journal of Advanced Manufacturing and Processing, Vol: 1, ISSN: 2637-403X
  • Journal article
    Nikolados E, Weisse A, Ceroni F, Oyarzun Det al., 2019,

    Growth defects and loss-of-function in synthetic gene circuits

    , ACS Synthetic Biology, Vol: 8, Pages: 1231-1240, ISSN: 2161-5063

    Synthetic gene circuits perturb the physiology of their cellular host. The extra load on endogenous processes shifts the equilibrium of resource allocation in the host, leading to slow growth and reduced biosynthesis. Here we built integrated host-circuit models to quantify growth defects caused by synthetic gene circuits. Simulations reveal a complex relation between circuit output and cellular capacity for gene expression. For weak induction of heterologous genes, protein output can be increased at the expense of growth defects. Yet for stronger induction, cellular capacity reaches a tipping point, beyond which both gene expression and growth rate drop sharply. Extensive simulations across various growth conditions and large regions of the design space suggest that the critical capacity is a result of ribosomal scarcity. We studied the impact of growth defects on various gene circuits and transcriptional logic gates, which highlights the extent to which cellular burden can limit, shape, and even break down circuit function. Our approach offers a comprehensive framework to assess the impact of host-circuit interactions in silico, with wide-ranging implications for the design and optimization of bacterial gene circuits.

  • Journal article
    Brittain R, Jones N, Ouldridge T, 2019,

    Biochemical Szilard engines for memory-limited inference

    , New Journal of Physics, Vol: 21, ISSN: 1367-2630

    By designing and leveraging an explicit molecular realisation of a measurement-and-feedback-powered Szilard engine, we investigate the extraction of work from complex environments by minimal machines with finite capacity for memory and decision-making. Living systems perform inference to exploit complex structure, or correlations, in their environment, but the physical limits and underlying cost/benefit trade-offs involved in doing so remain unclear. To probe these questions, we consider a minimal model for a structured environment—a correlated sequence of molecules—and explore mechanisms based on extended Szilard engines for extracting the work stored in these non-equilibrium correlations. We consider systems limited to a single bit of memory making binary 'choices' at each step. We demonstrate that increasingly complex environments allow increasingly sophisticated inference strategies to extract more free energy than simpler alternatives, and argue that optimal design of such machines should also consider the free energy reserves required to ensure robustness against fluctuations due to mistakes.

  • Software
    Quast N, Ouldridge T, 2019,

    Simulation of DNA tile self-assembly

    Submitted in accompaniment with final year report of MEng Project. DNA tile self-assembly is the spontaneous assembly of nano-structures made from short single-stranded DNA sequences. Successful assembly occurs within a narrow parameter window. Thisproject presents a model with which DNA self-assembly is simulated. Simulations for different tem-perature, sequence binding specificity and DNA tile concentrations indicate that: the growth rateof assemblies from uniform strand solutions is linear and highly temperature dependent; the aver-age nucleation times of assembly increase exponentially with temperature; high binding strengthsof boundary strands improve the stability of the complete assembly; locally high concentrations ofstrands seed the growth of the assembly; and locally low strand concentrations spatially direct thegrowth of the assembly. The source code is written in C.

  • Journal article
    Varghese F, Kabasakal BV, Cotton CA, Schumacher J, Rutherford AW, Fantuzzi A, Murray JWet al., 2019,

    A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii

    , Journal of Biological Chemistry, Vol: 294, Pages: 9367-9376, ISSN: 0021-9258

    The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

  • Journal article
    Madsen C, Goni Moreno A, Palchick Z P U, Roehner N, Bartley B, Bhatia S, Bhakta S, Bissell M, Clancy K, Cox RS, Gorochowski T, Grunberg R, Luna A, McLaughlin J, Nguyen T, Le Novere N, Pocock M, Sauro H, Scott-Brown J, Sexton JT, Stan G-B, Tabor JJ, Voigt CA, Zundel Z, Myers C, Beal J, Wipat Aet al., 2019,

    Synthetic Biology Open Language Visual (SBOL Visual) version 2.1

    , Journal of Integrative Bioinformatics, Vol: 16, Pages: 1-78, ISSN: 1613-4516

    People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species . Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.1 of SBOL Visual, which builds on the prior SBOL Visual 2.0 standard by expanding diagram syntax to include methods for showing modular structure and mappings between elements of a system, interactions arrows that can split or join (with the glyph at the split or join indicating either superposition or a chemical process), and adding new glyphs for indicating genomic context (e.g., integration into a plasmid or genome) and for stop codons.

  • Journal article
    Kotidis P, Demis P, Goey C, Correa E, McIntosh C, Trepekli S, Shah N, Klymenko O, Kontoravdi Ket al., 2019,

    Constrained global sensitivity analysis for bioprocess design space identification

    , Computers and Chemical Engineering, Vol: 125, Pages: 558-568, ISSN: 1873-4375

    The manufacture of protein-based therapeutics presents unique challenges due to limited control over the biotic phase. This typically gives rise to a wide range of protein structures of varying safety and in vivo efficacy. Herein we propose a computational methodology, enabled by the application of constrained Global Sensitivity Analysis, for efficiently exploring the operatingrange of process inputs in silico and identifying a design space that meets output constraints. The methodology was applied to an antibody-producing Chinese hamster ovary (CHO) cell culture system: we explored >8000 feeding strategies to identify a subset of manufacturing conditions that meet constraints on antibody titre and glycan distribution as an attribute of product quality. Our computational findings were then verified experimentally, confirming the applicability of this approach to a challenging production system. We envisage that this methodology can significantly expedite bioprocess development and increase operational flexibility.

  • Journal article
    Det-Udom R, Gilbert C, Liu L, Prakitchaiwattana C, Ellis T, Ledesma Amaro Ret al., 2019,

    Towards semi-synthetic microbial communities: Enhancing soy sauce fermentation properties in B. subtilis co-cultures

    , Microbial Cell Factories, Vol: 18, ISSN: 1475-2859

    BackgroundMany fermented foods and beverages are produced through the action of complex microbial communities. Synthetic biology approaches offer the ability to genetically engineer these communities to improve the properties of these fermented foods. Soy sauce is a fermented condiment with a vast global market. Engineering members of the microbial communities responsible for soy sauce fermentation may therefore lead to the development of improved products. One important property is the colour of soy sauce, with recent evidence pointing to a consumer preference for more lightly-coloured soy sauce products for particular dishes.ResultsHere we show that a bacterial member of the natural soy sauce fermentation microbial community, Bacillus, can be engineered to reduce the ‘browning’ reaction during soy sauce production. We show that two approaches result in ‘de-browning’: engineered consumption of xylose, an important precursor in the browning reaction, and engineered degradation of melanoidins, the major brown pigments in soy sauce. Lastly, we show that these two strategies work synergistically using co-cultures to result in enhanced de-browning.ConclusionsOur results demonstrate the potential of using synthetic biology and metabolic engineering methods for fine-tuning the process of soy sauce fermentation and indeed for many other natural food and beverage fermentations for improved products.

  • Journal article
    Ellis T, 2019,

    What is synthetic genomics anyway?

    , The Biochemist, Vol: 41, Pages: 6-9, ISSN: 0954-982X

    You may have heard of synthetic genomics. This headline-grabbing, high-profile, big science topic is starting to emerge catalysed by the pioneering work of famous names in synthetic biology and biotechnology like George Church and Craig Venter. But what is synthetic genomics and what is it being used for? As a prominent researcher at a recent UK meeting said: “Is it just synthetic biology with bigger bits of DNA?” Well no, not quite…

  • 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
    Witmer K, Fraschka SAK, Vlachou D, Bártfai R, Christophides Get al., 2019,

    Epigenetic regulation underlying Plasmodium berghei gene expression during its developmental transition from host to vector

    , bioRxiv, ISSN: 2045-2322

    ABSTRACT Epigenetic regulation of gene expression is an important attribute in the survival and adaptation of the malaria parasite Plasmodium in its human host. Our understanding of epigenetic regulation of gene expression in Plasmodium developmental stages beyond asexual replication in the mammalian host is sparse. We used chromatin immune-precipitation (ChIP) and RNA sequencing to create an epigenetic and transcriptomic map of the murine parasite Plasmodium berghei development from asexual blood stages to male and female gametocytes, and finally, to ookinetes. We show that heterochromatin 1 (HP1) almost exclusively associates with variantly expressed gene families at subtelomeric regions and remains stable across stages and various parasite lines. Variant expression based on heterochromatic silencing is observed only in very few genes. In contrast, the active histone mark histone 3 Lysine 9 acetylation (H3K9ac) is found between heterochromatin boundaries and occurs as a sharp peak around the start codon for ribosomal protein genes. H3K9ac occupancy positively correlates with gene transcripts in asexual blood stages, male gametocytes and ookinetes. Interestingly, H3K9ac occupancy does not correlate with transcript abundance in female gametocytes. Finally, we identify novel DNA motifs upstream of ookinete-specific genes thought to be involved in transcriptional activation upon fertilization.

  • Journal article
    Perin G, Jones PR, 2019,

    Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels.

    , Current Opinion in Biotechnology, Vol: 57, Pages: 175-182, ISSN: 0958-1669

    Currently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality.

  • 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
    Gang S, Sharma S, Saraf M, Buck M, Schumacher Jet al., 2019,

    Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method

    , BIO-PROTOCOL, Vol: 9
  • 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
  • Journal article
    Shaw W, Yamauchi H, Mead J, Gowers G, Bell D, Oling D, Larsson N, Wigglesworth M, Ladds G, Ellis Tet al., 2019,

    Engineering a model cell for rational tuning of GPCR signaling

    , Cell, Vol: 177, Pages: 782-796.e27, ISSN: 0092-8674

    G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

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