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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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    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.

    Boo A, Ellis T, Stan G,

    Host-Aware Synthetic Biology

    , Current Opinion in Systems Biology
    Aw R, Polizzi KM, 2019,

    Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform

    , BIOTECHNOLOGY AND BIOENGINEERING, Vol: 116, Pages: 656-666, ISSN: 0006-3592
    Kylilis N, Riangrungroj P, Lai H-E, Salema V, Fernández LÁ, Stan G-BV, Freemont PS, Polizzi KMet al., 2019,

    Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications.

    , ACS Sens, Vol: 4, Pages: 370-378

    Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design to detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straightforward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanobodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites, as well as routine medical testing and personalized medicine.

    Shaw W, Yamauchi H, Mead J, Gowers G, Bell D, Oling D, Larsson N, Wigglesworth M, Ladds G, Ellis Tet al.,

    Engineering a model cell for rational tuning of GPCR signaling

    , Cell, ISSN: 0092-8674

    G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond tospecific cues in their environment. However, the relationship between stimulus and response for eachGPCR is difficult to predict due to diversity in natural signal transduction architecture and expression.Using genome engineering in yeast, we here constructed an insulated, modular GPCR signaltransduction system to study how the response to stimuli can be predictably tuned using synthetictools. We delineated the contributions of a minimal set of key components via computational andexperimental 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 engineeredto respond to desired concentrations of peptides, metabolites, and hormones relevant to humanhealth. This work enables rational tuning of cell sensing, while providing a framework to guidereprogramming of GPCR-based signaling in other systems.

    Poulton J, Wolde PRT, Ouldridge TE, 2019,

    Non-equilibrium correlations in minimal dynamical models of polymer copying

    , Proceedings of the National Academy of Sciences, Vol: 116, Pages: 1946-1951, ISSN: 0027-8424

    Living systems produce "persistent" copies of information-carrying polymers, in which template and copy sequences remain correlated after physically decoupling. We identify a general measure of the thermodynamic efficiency with which these non-equilibrium states are created, and analyze the accuracy and efficiency of a family of dynamical models that produce persistent copies. For the weakest chemical driving, when polymer growth occurs in equilibrium, both the copy accuracy and, more surprisingly, the efficiency vanish. At higher driving strengths, accuracy and efficiency both increase, with efficiency showing one or more peaks at moderate driving. Correlations generated within the copy sequence, as well as between template and copy, store additional free energy in the copied polymer and limit the single-site accuracy for a given chemical work input. Our results provide insight in the design of natural self-replicating systems and can aid the design of synthetic replicators.

    Kylilis N, Riangrungroj P, Lai H-E, Salema V, Angel Fernandez L, Stan G-BV, Freemont PS, Polizzi KMet al., 2019,

    Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications

    , ACS SENSORS, Vol: 4, Pages: 370-378, ISSN: 2379-3694
    Kuntz J, Thomas P, Stan G-B, Barahona Met al., 2019,

    The exit time finite state projection scheme: bounding exit distributions and occupation measures of continuous-time Markov chains

    We introduce the exit time finite state projection (ETFSP) scheme, atruncation-based method that yields approximations to the exit distribution andoccupation measure associated with the time of exit from a domain (i.e., thetime of first passage to the complement of the domain) of time-homogeneouscontinuous-time Markov chains. We prove that: (i) the computed approximationsbound the measures from below; (ii) the total variation distances between theapproximations and the measures decrease monotonically as states are added tothe truncation; and (iii) the scheme converges, in the sense that, as thetruncation tends to the entire state space, the total variation distances tendto zero. Furthermore, we give a computable bound on the total variationdistance between the exit distribution and its approximation, and we delineatethe cases in which the bound is sharp. We also revisit the related finite stateprojection scheme and give a comprehensive account of its theoreticalproperties. We demonstrate the use of the ETFSP scheme by applying it to twobiological examples: the computation of the first passage time associated withthe expression of a gene, and the fixation times of competing species subjectto demographic noise.

    Gilbert C, Ellis T, 2019,

    Biological Engineered Living Materials: Growing Functional Materials with Genetically Programmable Properties

    , ACS SYNTHETIC BIOLOGY, Vol: 8, Pages: 1-15, ISSN: 2161-5063
    Ellis T, 2019,

    Predicting how evolution will beat us

    , MICROBIAL BIOTECHNOLOGY, Vol: 12, Pages: 41-43, ISSN: 1751-7915
    Weenink T, van der Hilst J, McKiernan R, Ellis Tet al., 2019,

    Design of RNA hairpin modules that predictably tune translation in yeast

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    Modular parts for tuning translation are prevalent in prokaryotic synthetic biology but lacking for eukaryotic synthetic biology. Working in Saccharomyces cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5′ untranslated region (5′UTR) of mRNAs can be used to tune expression levels by 100-fold by inhibiting translation. We determine the relationship between the calculated free energy of folding in the 5′UTR and in vivo protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this new tool, computational RNA sequence design can be used to predictably fine-tune protein production for genes expressed in yeast.

    Blount B, Ellis T, 2019,

    The Synthetic Genome Summer Course

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    The Synthetic Genome Summer Course was convened with the aim of teaching a wide range of researchers the theory and practical skills behind recent advances in synthetic biology and synthetic genome science, with a focus on Sc2.0, the synthetic yeast genome project. Through software workshops, tutorials and research talks from leading members of the field, the 30 attendees learnt about relevant principles and techniques that they were then able to implement first-hand in laboratory-based practical sessions. Participants SCRaMbLEd semi-synthetic yeast strains to diversify heterologous pathways, used automation to build combinatorial pathway libraries and used CRISPR to debug fitness defects caused by synthetic chromosome design changes. Societal implications of synthetic chromosomes were explored and industrial stakeholders discussed synthetic biology from a commercial standpoint. Over the 5 days, participants gained valuable insight and acquired skills to aid them in future synthetic genome research.

    Ouldridge TE, Brittain RA, Wolde PRT, 2018,

    The power of being explicit: demystifying work, heat, and free energy in the physics of computation

    Interest in the thermodynamics of computation has revived in recent years,driven by developments in science, economics and technology. Given theconsequences of the growing demand for computational power, the idea ofreducing the energy cost of computations has gained new importance.Simultaneously, many biological networks are now interpreted asinformation-processing or computational systems constrained by their underlyingthermodynamics. Indeed, some suggest that low-cost, high-density biologicalsystems may help to mitigate the rising demand for computational power and the"end" of Moore's law of exponential growth in the density of transistors. In this chapter we address widespread misconceptions about thermodynamics andthe thermodynamics of computation. In particular, we will argue against thegeneral perception that a measurement or copy operation can be performed at nocost, against the emphasis placed on the significance of erasure operations,and against the careless discussion of heat and work. While not universal,these misconceptions are sufficiently prevalent (particularly withininterdisciplinary contexts) to warrant a detailed discussion. In the process,we will argue that explicitly representing fundamental processes is a usefultool, serving to demystify key concepts. We first give a brief overview of thermodynamics, then the history of thethermodynamics of computation - particularly in terms of copy and measurementoperations inherent to classic thought experiments. Subsequently, we analysethese ideas via an explicit biochemical representation of the entire cycle ofSzilard's engine. In doing so we show that molecular computation is both apromising engineering paradigm, and a valuable tool in providing fundamentalunderstanding.

    Walker KT, Goosens VJ, Das A, Graham AE, Ellis Tet al., 2018,

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

    , Microb Biotechnol

    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.

    Taylor GM, Mordaka PM, Heap JT, 2018,

    Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

    , Nucleic Acids Res

    DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

    Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, Nolan T, Crisanti Aet al., 2018,

    A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 1062-+, ISSN: 1087-0156
    Kelly CL, Harris AWK, Steel H, Hancock EJ, Heap JT, Papachristodoulou Aet al., 2018,

    Synthetic negative feedback circuits using engineered small RNAs

    , NUCLEIC ACIDS RESEARCH, Vol: 46, Pages: 9875-9889, ISSN: 0305-1048
    Trantidou T, Dekker L, Polizzi K, Ces O, Elani Yet al., 2018,

    Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

    , INTERFACE FOCUS, Vol: 8, ISSN: 2042-8898
    Gorochowski TE, Ellis T, 2018,

    Designing efficient translation

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 934-935, ISSN: 1087-0156
    Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018,

    Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

    , SCIENTIFIC REPORTS, Vol: 8, ISSN: 2045-2322

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