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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 articleTaylor G, Mordaka P, Heap J, 2019,
Start-stop assembly: a functionally scarless DNA assembly system optimized for metabolic engineering
, Nucleic Acids Research, Vol: 47, Pages: e17-e17, ISSN: 0305-1048DNA 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 minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.
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Conference paperGirvan P, Teng X, Brooks NJ, et al., 2019,
Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications
, 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495 -
Journal articlePoulton 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: 0027-8424Living 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.
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Journal articleMcCarty NS, Ledesma-Amaro R, 2019,
Synthetic biology tools to engineer microbial communities for biotechnology
, Trends in Biotechnology, Vol: 37, Pages: 181-197, ISSN: 0167-7799Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial consortia for diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and the development of new tools to construct synthetic consortia and program their behaviors, will vastly expand the functions that can be performed by communities of interacting microorganisms. Here, we review recent advancements in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss ongoing and emerging efforts to apply consortia for various biotechnological applications, and suggest future applications.
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Conference paperSchumacher J, Waite C, Wang B, 2019,
Synthetic transcription factors allowtuneable synthetic control of the complex bacterial nor regulon
, EMBO: Creating is Understanding: Synthetic Biology Masters Complexity -
Journal articleFriddin M, Bolognesi G, Salehi-Reyhani A, et al., 2019,
Direct manipulation of liquid ordered lipid membrane domains using optical traps
, Communications Chemistry, Vol: 2, Pages: 1-7, ISSN: 2399-3669Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning.
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Journal articleHabtewold T, Tapanelli S, Ellen KG M, et al., 2019,
Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity
, Malaria Journal, Vol: 18, ISSN: 1475-2875BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.
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Journal articleTosi T, Hoshiga F, Millership C, et al., 2019,
Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM
, PLoS Pathogens, Vol: 15, Pages: 1-28, ISSN: 1553-7366c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
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Journal articleGilbert C, Ellis T, 2019,
Biological engineered living materials - growing functional materials with genetically-programmable properties
, ACS Synthetic Biology, Vol: 8, Pages: 1-15, ISSN: 2161-5063Natural biological materials exhibit remarkable properties: self-assembly from simple raw materials, precise control of morphology, diverse physical and chemical properties, self-repair and the ability to sense-and-respond to environmental stimuli. Despite having found numerous uses in human industry and society, the utility of natural biological materials is limited. But, could it be possible to genetically program microbes to create entirely new and useful biological materials? At the intersection between microbiology, material science and synthetic biology, the emerging field of biological Engineered Living Materials (ELMs) aims to answer this question. Here we review recent efforts to program cells to produce living materials with novel functional properties, focussing on microbial systems that can be engineered to grow materials and on new genetic circuits for pattern formation that could be used to produce the more complex systems of the future.
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Journal articleExley K, Reynolds C, Suckling L, et al., 2019,
Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway
, Journal of Biological Engineering, Vol: 13, ISSN: 1754-1611BackgroundThe automation of modular cloning methodologies permits the assembly of many genetic designs. Utilising characterised biological parts aids in the design and redesign of genetic pathways. The characterisation information held on datasheets can be used to determine whether a biological part meets the design requirements. To manage the design of genetic pathways, researchers have turned to modelling-based computer aided design software tools.ResultAn automated workflow has been developed for the design and build of heterologous metabolic pathways. In addition, to demonstrate the powers of electronic datasheets we have developed software which can transfer part information from a datasheet to the Design of Experiment software JMP. To this end we were able to use Design of Experiment software to rationally design and test randomised samples from the design space of a lycopene pathway in E. coli. This pathway was optimised by individually modulating the promoter strength, RBS strength, and gene order targets.ConclusionThe use of standardised and characterised biological parts will empower a design-oriented synthetic biology for the forward engineering of heterologous expression systems. A Design of Experiment approach streamlines the design-build-test cycle to achieve optimised solutions in biodesign. Developed automated workflows provide effective transfer of information between characterised information (in the form of datasheets) and DoE software.
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Conference paperAppuswamy R, Brigand KL, Barbry P, et al., 2019,
OligoArchive: Using DNA in the DBMS storage hierarchy
, CIDR 2019 -
Journal articleKis Z, Shattock R, Shah N, et al., 2019,
Emerging technologies for low-cost, rapid vaccine manufacture
, Biotechnology Journal, Vol: 14, ISSN: 1860-6768To stop the spread of future epidemics and meet infant vaccination demands in low‐ and middle‐income countries, flexible, rapid and low‐cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high‐yield yeast recombinant protein vaccines; b) insect cell‐baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale‐up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.
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Journal articleNash A, Urdaneta Mignini G, Beaghton A, et al., 2019,
Integral Gene Drives for population replacement
, Biology Open, Vol: 8, ISSN: 2046-6390A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.
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Journal articleGu Y, Lv X, Liu Y, et al., 2019,
Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis
, Metabolic Engineering, Vol: 51, Pages: 59-69, ISSN: 1096-7176One of the primary goals of microbial metabolic engineering is to achieve high titer, yield and productivity (TYP) of engineered strains. This TYP index requires optimized carbon flux toward desired molecule with minimal by-product formation. De novo redesign of central carbon and redox metabolism holds great promise to alleviate pathway bottleneck and improve carbon and energy utilization efficiency. The engineered strain, with the overexpression or deletion of multiple genes, typically can’t meet the TYP index, due to overflow of central carbon and redox metabolism that compromise the final yield, despite a high titer or productivity might be achieved. To solve this challenge, we reprogramed the central carbon and redox metabolism of Bacillus subtilis and achieved high TYP production of N-acetylglucosamine. Specifically, a “push–pull–promote” approach efficiently reduced the overflown acetyl-CoA flux and eliminated byproduct formation. Four synthetic NAD(P)-independent metabolic routes were introduced to rewire the redox metabolism to minimize energy loss. Implementation of these genetic strategies led us to obtain a B. subtilis strain with superior TYP index. GlcNAc titer in shake flask was increased from 6.6 g L−1 to 24.5 g L−1, the yield was improved from 0.115 to 0.468 g GlcNAc g−1 glucose, and the productivity was increased from 0.274 to 0.437 g L−1 h−1. These titer and yield are the highest levels ever reported and, the yield reached 98% of the theoretical pathway yield (0.478 g g−1 glucose). The synthetic redesign of carbon metabolism and redox metabolism represent a novel and general metabolic engineering strategy to improve the performance of microbial cell factories.
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Conference paperAppuswamy R, Lebrigand K, Barbry P, et al., 2019,
Oligoarchive: Using DNA in the DBMS storage hierarchy
The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
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Conference paperWebb AJ, Landeryou T, Kelwick R, et al., 2019,
SPECIFIC NUCLEIC ACIDS LIGATION FOR DETECTION OF SCHISTOSOMES: SNAILS
, 68th Annual Meeting of the American-Society-for-Tropical-Medicine-and-Hygiene (ASTMH), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 182-182, ISSN: 0002-9637 -
Journal articleWeenink T, van der Hilst J, McKiernan R, et al., 2019,
Design of RNA hairpin modules that predictably tune translation in yeast
, Synthetic Biology, Vol: 3, ISSN: 2397-7000Modular 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.
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Journal articleEyerich K, Brown S, Perez White B, et al., 2019,
Human and computational models of atopic dermatitis: a review and perspectives by an expert panel of the International Eczema Council
, Journal of Allergy and Clinical Immunology, Vol: 143, Pages: 36-45, ISSN: 0091-6749Atopic dermatitis (AD) is a prevalent disease worldwide and is associated with systemic comorbidities representing a significant burden on patients, their families, and society. Therapeutic options for AD remain limited, in part because of a lack of well-characterized animal models. There has been increasing interest in developing experimental approaches to study the pathogenesis of human AD in vivo, in vitro, and in silico to better define pathophysiologic mechanisms and identify novel therapeutic targets and biomarkers that predict therapeutic response. This review critically appraises a range of models, including genetic mutations relevant to AD, experimental challenge of human skin in vivo, tissue culture models, integration of “omics” data sets, and development of predictive computational models. Although no one individual model recapitulates the complex AD pathophysiology, our review highlights insights gained into key elements of cutaneous biology, molecular pathways, and therapeutic target identification through each approach. Recent developments in computational analysis, including application of machine learning and a systems approach to data integration and predictive modeling, highlight the applicability of these methods to AD subclassification (endotyping), therapy development, and precision medicine. Such predictive modeling will highlight knowledge gaps, further inform refinement of biological models, and support new experimental and systems approaches to AD.
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Journal articleBlount B, Ellis T, 2019,
The Synthetic Genome Summer Course
, Synthetic Biology, Vol: 3, ISSN: 2397-7000The 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.
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Conference paperAppuswamy R, Lebrigand K, Barbry P, et al., 2019,
Oligoarchive: Using DNA in the DBMS storage hierarchy
© 2019 Conference on Innovative Data Systems Research (CIDR). All rights reserved. The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
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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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