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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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  • Book chapter
    Lai H-E, Moore S, Polizzi K, Freemont Pet al., 2018,

    EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

    , Pages: 429-444

    Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

  • Journal article
    Khara DC, Schreck JS, Tomov TE, Berger Y, Ouldridge TE, Doye JPK, Nir Eet al., 2017,

    DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size

    , Nucleic Acids Research, Vol: 46, Pages: 1553-1561, ISSN: 0305-1048

    We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions.

  • Journal article
    Broedel AK, Isalan M, Jaramillo A, 2017,

    Engineering of biomolecules by bacteriophage directed evolution

    , Current Opinion in Biotechnology, Vol: 51, Pages: 32-38, ISSN: 0958-1669

    Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein–protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes.

  • Journal article
    Ouldridge TE, 2017,

    The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamics

    , Natural Computing, Vol: 17, Pages: 3-29, ISSN: 1567-7818

    Improved understanding of molecular systems has only emphasised thesophistication of networks within the cell. Simultaneously, the advance ofnucleic acid nanotechnology, a platform within which reactions can beexquisitely controlled, has made the development of artificial architecturesand devices possible. Vital to this progress has been a solid foundation in thethermodynamics of molecular systems. In this pedagogical review andperspective, I will discuss how thermodynamics determines both the overallpotential of molecular networks, and the minute details of design. I will thenargue that, in turn, the need to understand molecular systems is helping todrive the development of theories of thermodynamics at the microscopic scale.

  • Journal article
    Royle KE, Polizzi KM, 2017,

    A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.

  • Journal article
    Wintle BC, Boehm CR, Rhodes C, Molloy JC, Millett P, Adam L, Breitling R, Carlson R, Casagrande R, Dando M, Doubleday R, Drexler E, Edwards B, Ellis T, Evans NG, Hammond R, Haseloff J, Kahl L, Kuiken T, Lichman BR, Matthewman CA, Napier JA, Oheigeartaigh SS, Patron NJ, Perello E, Shapira P, Tait J, Takano E, Sutherland WJet al., 2017,

    A transatlantic perspective on 20 emerging issues in biological engineering

    , eLife, Vol: 6, ISSN: 2050-084X

    Advances in biological engineering are likely to have substantial impacts on global society. To explorethese potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunitiesand risks presented by biological engineering. We first identified 70 potential issues, and then used an iterativeprocess to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to berelatively unknown outside the field of biological engineering. The issues identified may be of interest toresearchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.

  • Journal article
    Park YK, Nicaud JM, Ledesma Amaro R, 2017,

    The engineering potential of Rhodosporidium toruloides as a workhorse for biotechnological applications

    , Trends in Biotechnology, Vol: 36, Pages: 304-317, ISSN: 0167-7799

    Moving our society towards a bioeconomy requires efficient and sustainable microbial production of chemicals and fuels. Rhodotorula (Rhodosporidium) toruloides is a yeast that naturally synthesizes substantial amounts of specialty chemicals and has been recently engineered to (i) enhance its natural production of lipids and carotenoids, and (ii) produce novel industrially relevant compounds. The use of R. toruloides by companies and research groups has exponentially increased in recent years as a result of recent improvements in genetic engineering techniques and the availability of multiomics information on its genome and metabolism. This review focuses on recent engineering approaches in R. toruloides for bioproduction and explores its potential as a biotechnological chassis.

  • Journal article
    Selles Vidal L, Kelly CL, Mordaka PM, Heap JTet al., 2017,

    Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application.

    , Biochimica et Biophysica Acta - Proteins and Proteomics, Vol: 1866, Pages: 327-347, ISSN: 1570-9639

    NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)(+). NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilisation, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

  • Journal article
    Deshpande A, Ouldridge TE, 2017,

    High rates of fuel consumption are not required by insulating motifs to suppress retroactivity in biochemical circuits

    , Engineering Biology, Vol: 1, Pages: 86-99, ISSN: 2398-6182

    Retroactivity arises when the coupling of a molecular network $\mathcal{U}$to a downstream network $\mathcal{D}$ results in signal propagation back from$\mathcal{D}$ to $\mathcal{U}$. The phenomenon represents a breakdown inmodularity of biochemical circuits and hampers the rational design of complexfunctional networks. Considering simple models of signal-transductionarchitectures, we demonstrate the strong dependence of retroactivity on theproperties of the upstream system, and explore the cost and efficacy offuel-consuming insulating motifs that can mitigate retroactive effects. We findthat simple insulating motifs can suppress retroactivity at a low fuel cost bycoupling only weakly to the upstream system $\mathcal{U}$. However, this designapproach reduces the signalling network's robustness to perturbations from leakreactions, and potentially compromises its ability to respond torapidly-varying signals.

  • Journal article
    Larroude M, Celinska E, Back A, Thomas S, Nicaud JM, Ledesma Amaro Ret al., 2017,

    A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β-carotene

    , Biotechnology and Bioengineering, Vol: 115, Pages: 464-472, ISSN: 1097-0290

    The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene.

  • Journal article
    Ogonah OW, Polizzi KM, Bracewell DG, 2017,

    Cell free protein synthesis: a viable option for stratified medicines manufacturing?

    , CURRENT OPINION IN CHEMICAL ENGINEERING, Vol: 18, Pages: 77-83, ISSN: 2211-3398
  • Journal article
    Hancock E, Ang J, Papachristodoulou A, Stan Get al., 2017,

    The interplay between feedback and buffering in homeostasis

    , Cell Systems, Vol: 5, Pages: 498-508.e23, ISSN: 2405-4720

    Feedback and buffering---the use of reservoirs of molecules to maintain molecular concentrations---are the primary mechanisms for robust regulation in biochemical processes. The universal principles behind their combined effect, however, have not been studied before. Here, we determine the fundamental forms of cooperation and tradeoff between buffering and feedback. We find that negative feedback regulates slow-changing components of time-varying signals, while buffering regulates fast-changing components, consistent with observations of both ATP and pH regulation. We further find that buffering stabilizes feedback and improves its effectiveness, but also introduces molecular noise. In addition, we show that rapid-acting buffering imparts negative derivative feedback, while slow-acting buffering can result in feedforward filtering of specific signals; both are control strategies widely used in technology. Finally, we discover an empirical cross-species relationship between feedback in glycolysis and ATP buffering that is consistent with our findings.

  • Journal article
    Mitchell LA, Ellis T, 2017,

    Synthetic genome engineering gets infectious

    , PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 114, Pages: 11006-11008, ISSN: 0027-8424
  • Journal article
    Wen KY, Cameron L, Chappell J, Jensen K, Bell DJ, Kelwick R, Kopniczky M, Davies JC, Filloux A, Freemont PSet al., 2017,

    A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples.

    , ACS Synthetic Biology, Vol: 6, Pages: 2293-2301, ISSN: 2161-5063

    Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

  • Journal article
    Hammond AM, Kyrou K, Bruttini M, North A, Galizi R, Karlsson X, Kranjc N, Carpi FM, D'Aurizio R, Crisanti A, Nolan Tet al., 2017,

    The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito.

    , PLoS Genetics, Vol: 13, ISSN: 1553-7390

    Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.

  • Journal article
    Jewett MC, Ellis T, 2017,

    Editorial overview: Synthetic biology: Frontiers in synthetic biology

    , CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 40, Pages: A1-A3, ISSN: 1367-5931
  • Journal article
    Sou SN, Ken L, Nayyar K, Polizzi KM, Sellick C, Kontoravdi Cet al., 2017,

    Exploring cellular behaviour under transient geneexpression and its impact on mAb productivity and Fc glycosylation

    , Biotechnology and Bioengineering, Vol: 115, Pages: 512-518, ISSN: 1097-0290

    Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently-expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in qP compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources towards mAb synthesis and Fc-glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N-acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi-antennary glycan structures for further processing.

  • Journal article
    Werther R, Hallinan JP, Lambert AR, Havens K, Pogson M, Jarjour J, Galizi R, Windbichler N, Crisanti A, Nolan T, Stoddard BLet al., 2017,

    Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

    , Nucleic Acids Research, Vol: 45, Pages: 8621-8634, ISSN: 0305-1048

    The retargeting of protein–DNA specificity, outsideof extremely modular DNA binding proteins suchas TAL effectors, has generally proved to be quitechallenging. Here, we describe structural analysesof five different extensively retargeted variants of asingle homing endonuclease, that have been shownto function efficiently in ex vivo and in vivo applications.The redesigned proteins harbor mutationsat up to 53 residues (18%) of their amino acid sequence,primarily distributed across the DNA bindingsurface, making them among the most signifi-cantly reengineered ligand-binding proteins to date.Specificity is derived from the combined contributionsof DNA-contacting residues and of neighboringresidues that influence local structural organization.Changes in specificity are facilitated by theability of all those residues to readily exchange bothform and function. The fidelity of recognition is notprecisely correlated with the fraction or total numberof residues in the protein–DNA interface that areactually involved in DNA contacts, including directionalhydrogen bonds. The plasticity of the DNArecognitionsurface of this protein, which allows substantialretargeting of recognition specificity withoutrequiring significant alteration of the surroundingprotein architecture, reflects the ability of the correspondinggenetic elements to maintain mobility andpersistence in the face of genetic drift within potentialhost target sites.

  • Journal article
    Broedel AK, Jaramillo A, Isalan M, 2017,

    Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

    , Nature Protocols, Vol: 12, Pages: 1830-1843, ISSN: 1750-2799

    Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene—or gene circuit function—that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.

  • Journal article
    Mannan AA, Liu D, Zhang F, Oyarzun DAet al., 2017,

    Fundamental design principles for transcription-factor-based metabolite biosensors

    , ACS Synthetic Biology, Vol: 6, Pages: 1851-1859, ISSN: 2161-5063

    Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose–response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.

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