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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 articleHazel P, Kroll SHB, Bondke A, et al., 2018,
Corrigendum: Inhibitor selectivity for cyclin-dependent kinase 7: a structural, thermodynamic, and modelling study
, ChemMedChem, Vol: 13, Pages: 207-207, ISSN: 1860-7187 -
Journal articleFreemont PS, Salih O, He S, et al., 2018,
Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa
, Structure, Vol: 26, Pages: 329-336.e3, ISSN: 0969-2126Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
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Book chapterLai H-E, Moore S, Polizzi K, et al., 2018,
EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.
, Pages: 429-444Development 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.
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Journal articleKhara DC, Schreck JS, Tomov TE, et 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: 0305-1048We 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.
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Journal articleKreula SM, Kaewphan S, Ginter F, et al., 2017,
Finding novel relationships with integrated gene-gene association network analysis of <i>Synechocystis sp. </i>PCC 6803 using species-independent text-mining
<jats:p>The increasing move towards open access full-text scientific literature enhances our ability to utilize advanced text-mining methods to construct information-rich networks that no human will be able to grasp simply from 'reading the literature'. The utility of text-mining for well-studied species is obvious though the utility for less studied species, or those with no prior track-record at all, is not clear. Here we present a concept for how advanced text-mining can be used to create information-rich networks even for less well studied species and apply it to generate an open-access gene-gene association network resource for <jats:italic>Synechocystis sp.</jats:italic> PCC 6803, a representative model organism for cyanobacteria and first case-study for the methodology. By merging the text-mining network with networks generated from species-specific experimental data, network integration was used to enhance the accuracy of predicting novel interactions that are biologically relevant. A rule-based algorithm was constructed in order to automate the search for novel candidate genes with a high degree of likely association to known target genes by (1) ignoring established relationships from the existing literature, as they are already 'known', and (2) demanding multiple independent evidences for every novel and potentially relevant relationship. Using selected case studies, we demonstrate the utility of the network resource and rule-based algorithm to (<jats:italic>i</jats:italic>) discover novel candidate associations between different genes or proteins in the network, and (<jats:italic>ii</jats:italic>) rapidly evaluate the potential role of any one particular gene or protein. The full network is provided as an open source resource.</jats:p>
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Journal articleClaesen S, Stone A, van Rossum M, et al., 2017,
Comprehensive web-based broker for bio-technology design and manufacturing
, Engineering Biology, Vol: 1, Pages: 100-102, ISSN: 2398-6182Synthetic biology, particularly in relation to characterisation experiments relating to the description of bio-parts frequently involves the use of a wide range of equipment, including, for example, plate reader's, flow cytometers, and mass spectrometers. This equipment is often from multiple manufacturers. The study describes broker technology that has been developed which has the ability to connect multiple types of equipment into a common information environment; the connectivity from the databases and equipment is achieved using Visbion's ‘cube’ technology that involves military specification encryption for data security. The broker technology uses a new, developing standard, Digital Imaging and Communication in Medicine (DICOM)-SB, that is based on the highly successful international standard for biomedicine, DICOM. The broker uses a version of the DICOM data model that has been specifically designed for synthetic biology and, in particular, characterisation data.
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Journal articleDavidchack RL, Ouldridge TE, Tretyakov MV, 2017,
Geometric integrator for Langevin systems with quaternion-based rotational degrees of freedom and hydrodynamic interactions
, Journal of Chemical Physics, Vol: 147, ISSN: 0021-9606We introduce new Langevin-type equations describing the rotational andtranslational motion of rigid bodies interacting through conservative andnon-conservative forces, and hydrodynamic coupling. In the absence ofnon-conservative forces the Langevin-type equations sample from the canonicalensemble. The rotational degrees of freedom are described using quaternions,the lengths of which are exactly preserved by the stochastic dynamics. For theproposed Langevin-type equations, we construct a weak 2nd order geometricintegrator which preserves the main geometric features of the continuousdynamics. A number of numerical experiments are presented to illustrate boththe new Langevin model and the numerical method for it.
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Journal articleHancock E, Ang J, Papachristodoulou A, et al., 2017,
The interplay between feedback and buffering in homeostasis
, Cell Systems, Vol: 5, Pages: 498-508.e23, ISSN: 2405-4712Feedback 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.
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Journal articleBroedel AK, Isalan M, Jaramillo A, 2017,
Engineering of biomolecules by bacteriophage directed evolution
, Current Opinion in Biotechnology, Vol: 51, Pages: 32-38, ISSN: 0958-1669Conventional 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.
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Journal articleOuldridge 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: 1567-7818Improved 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.
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Journal articleRoyle KE, Polizzi KM, 2017,
A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris
, Scientific Reports, Vol: 7, ISSN: 2045-2322Although 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.
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Journal articleWintle BC, Boehm CR, Rhodes C, et al., 2017,
A transatlantic perspective on 20 emerging issues in biological engineering
, eLife, Vol: 6, ISSN: 2050-084XAdvances 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.
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Journal articlePark 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: 0167-7799Moving 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.
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Journal articleDeshpande 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: 2398-6182Retroactivity 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.
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Journal articleLarroude M, Celinska E, Back A, et 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: 1097-0290The 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.
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Journal articleOgonah 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 articleMitchell 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 articleWen KY, Cameron L, Chappell J, et 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: 2161-5063Synthetic 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.
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Journal articleHammond AM, Kyrou K, Bruttini M, et 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: 1553-7390Gene 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.
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Journal articleJewett 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
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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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