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Synthetic Biology underpins advances in the bioeconomy

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology. 

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.


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  • Journal article
    Kelly CL, Liu Z, Yoshihara A, Jenkinson SF, Wormald MR, Otero J, Estévez A, Kato A, Marqvorsen MHS, Fleet GWJ, Estévez RJ, Izumori K, Heap JTet al., 2016,

    Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.

    , ACS Synth Biol, Vol: 5, Pages: 1136-1145

    External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

  • Journal article
    Ellis T, Cai Y, 2016,

    Synthetic Biology in Europe

    , ACS SYNTHETIC BIOLOGY, Vol: 5, Pages: 1033-1033, ISSN: 2161-5063
  • Journal article
    Moore SJ, lai H-E, Kelwick R, Mei S, Bell DJ, Polizzi K, Freemont PSet al., 2016,

    EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology

    , ACS Synthetic Biology, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063

    Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.

  • Journal article
    MacDonald J, Freemont PS, 2016,

    Computational protein design with backbone plasticity

    , Biochemical Society Transactions, Vol: 44, Pages: 1523-1529, ISSN: 1470-8752

    The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as ‘scaffolds’ onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increased search space, but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process.

  • Journal article
    Clarke LJ, Kitney RI, 2016,

    Synthetic biology in the UK – An outline of plans and progress

    , Synthetic and Systems Biotechnology, Vol: 1, Pages: 243-257, ISSN: 2405-805X

    Synthetic biology is capable of delivering new solutions to key challenges spanning the bioeconomy, both nationally and internationally. Recognising this significant potential and the associated need to facilitate its translation and commercialisation the UK government commissioned the production of a national Synthetic Biology Roadmap in 2011, and subsequently provided crucial support to assist its implementation.Critical infrastructural investments have been made, and important strides made towards the development of an effectively connected community of practitioners and interest groups. A number of Synthetic Biology Research Centres, DNA Synthesis Foundries, a Centre for Doctoral Training, and an Innovation Knowledge Centre have been established, creating a nationally distributed and integrated network of complementary facilities and expertise.The UK Synthetic Biology Leadership Council published a UK Synthetic Biology Strategic Plan in 2016, increasing focus on the processes of translation and commercialisation. Over 50 start-ups, SMEs and larger companies are actively engaged in synthetic biology in the UK, and inward investments are starting to flow.Together these initiatives provide an important foundation for stimulating innovation, actively contributing to international research and development partnerships, and helping deliver useful benefits from synthetic biology in response to local and global needs and challenges.

  • Journal article
    Kelwick RJR, Webb AJ, MacDonald JT, Freemont PSet al., 2016,

    Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

    , Metabolic Engineering, Vol: 38, Pages: 370-381, ISSN: 1096-7184

    Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

  • Journal article
    Storch M, Casini A, Mackrow B, Ellis T, Baldwin GSet al., 2016,

    BASIC: A Simple and Accurate Modular DNA Assembly Method.

    , Methods Mol Biol, Vol: 1472, Pages: 79-91

    Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].

  • Journal article
    Awan A, Blount B, Bell D, Ho J, McKiernan R, Ellis Tet al., 2016,

    Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast

    , Biorxiv

    Abstract Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. By taking genes from a filamentous fungus and directing their efficient expression and subcellular localisation, we here engineer the baker’s yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide. Using synthetic biology tools combined with long-read DNA sequencing, we optimise productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

  • Journal article
    MacDonald JT, Kabasakal BV, Godding D, Kraatz S, Henderson L, Barber J, Freemont PS, Murray JWet al., 2016,

    Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 10346-10351, ISSN: 1091-6490

    The ability to design and construct structures with atomic level precisionis one of the key goals of nanotechnology. Proteins offer anattractive target for atomic design, as they can be synthesized chemicallyor biologically, and can self-assemble. However the generalizedprotein folding and design problem is unsolved. One approach tosimplifying the problem is to use a repetitive protein as a scaffold.Repeat proteins are intrinsically modular, and their folding and structuresare better understood than large globular domains. Here, wehave developed a new class of synthetic repeat protein, based onthe pentapeptide repeat family of beta-solenoid proteins. We haveconstructed length variants of the basic scaffold, and computationallydesigned de novo loops projecting from the scaffold core. Theexperimentally solved 3.56 ˚A resolution crystal structure of one designedloop matches closely the designed hairpin structure, showingthe computational design of a backbone extension onto a syntheticprotein core without the use of backbone fragments from knownstructures. Two other loop designs were not clearly resolved in thecrystal structures and one loop appeared to be in an incorrect conformation.We have also shown that the repeat unit can accommodatewhole domain insertions by inserting a domain into one of the designedloops.

  • Journal article
    Agustín-Pavón C, Mielcarek M, Garriga-Canut M, Isalan Met al., 2016,

    Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

    , Molecular Neurodegeneration, Vol: 11, Pages: 1-16, ISSN: 1750-1326

    Background: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequencesand are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZFKOX1)able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which arefound in Huntington’s disease (HD). This ZF acutely repressed mutant HTT expression in amouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In thepresent work, we sought to develop a long-term single-injection gene therapy approach in thebrain.Method: Since non-self proteins can elicit immune and inflammatory responses, we designed ahost-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely incombination with rAAV vector delivery. We also tested a neuron-specific enolase promoter(pNSE), which has been reported as enabling long-term transgene expression, to see whetherHTT repression could be observed for up to 6 months after AAV injection in the brain.Results: After rAAV vector delivery, we found that non-self proteins induce significantinflammatory responses in the brain, in agreement with previous studies. Specifically, microglialcells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP,respectively, and this was accompanied by a moderate neuronal loss. In contrast, the hostmatchedmZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAGpromoter (CMV early enhancer element and the chicken β-actin promoter) led to a strongreduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viralpromoter to see whether the host-adapted ZF expression could be sustained for a longer time.Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat)resulted in up to 77% repression of mutant HTT in whole brain, 3 weeks after bilateralintraventricular injection of 1010 virions. Importantly, repressions of 48% and 23% were stilldetected after 12 and 24 weeks

  • Journal article
    Toczek M, Zielonka D, Zukowska P, Marcinkowski JT, Slominska E, Isalan M, Smolenski RT, Mielcarek Met al., 2016,

    An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington's disease related cardiomyopathy

    , BBA - Molecular Basis of Disease, Vol: 1862, Pages: 2147-2157, ISSN: 0925-4439

    Huntington's disease (HD) is mainly thought of as a neurological disease, but multiple epidemiological studies havedemonstrated a number of cardiovascular events leading to heart failure in HD patients. Our recent studies showed anincreased risk of heart contractile dysfunction and dilated cardiomyopathy in HD pre-clinical models. This could potentiallyinvolve metabolic remodeling, that is a typical feature of the failing heart, with reduced activities of high energyphosphate generating pathways. In this study, we sought to identify metabolic abnormalities leading to HD-related cardiomyopathyin pre-clinical and clinical settings. We found that HD mouse models developed a profound deteriorationin cardiac energy equilibrium, despite AMP-activated protein kinase hyperphosphorylation. This was accompanied by areduced glucose usage and a significant deregulation of genes involved in de novo purine biosynthesis, in conversion ofadenine nucleotides, and in adenosine metabolism. Consequently, we observed increased levels of nucleotide catabolitessuch as inosine, hypoxanthine, xanthine and uric acid, in murine and human HD serum. These effects may be causedlocally by mutant HTT, via gain or loss of function effects, or distally by a lack of trophic signals from central nerve stimulation.Either may lead to energy equilibrium imbalances in cardiac cells, with activation of nucleotide catabolism plusan inhibition of re-synthesis. Our study suggests that future therapies should target cardiac mitochondrial dysfunction toameliorate energetic dysfunction. Importantly, we describe the first set of biomarkers related to heart and skeletal muscledysfunction in both pre-clinical and clinical HD settings.

  • Journal article
    Ceroni F, Blount BA, Ellis T, 2016,

    Sensing the Right Time to Be Productive

    , Cell Systems, Vol: 3, Pages: 116-117, ISSN: 2405-4720

    Engineered E. coli can be made to autonomously switch from growth to production by a modular two-gate system that reduces the burden of biosynthesis.

  • Journal article
    Sainz de Murieta I, Bultelle M, Kitney RI, 2016,

    Toward the first data acquisition standard in synthetic biology

    , ACS Synthetic Biology, Vol: 5, Pages: 817-826, ISSN: 2161-5063

    This paper describes the development of a new data acquisition standard for synthetic biology. This comprises the creation of a methodology that is designed to capture all the data, metadata, and protocol information associated with biopart characterization experiments. The new standard, called DICOM-SB, is based on the highly successful Digital Imaging and Communications in Medicine (DICOM) standard in medicine. A data model is described which has been specifically developed for synthetic biology. The model is a modular, extensible data model for the experimental process, which can optimize data storage for large amounts of data. DICOM-SB also includes services orientated toward the automatic exchange of data and information between modalities and repositories. DICOM-SB has been developed in the context of systematic design in synthetic biology, which is based on the engineering principles of modularity, standardization, and characterization. The systematic design approach utilizes the design, build, test, and learn design cycle paradigm. DICOM-SB has been designed to be compatible with and complementary to other standards in synthetic biology, including SBOL. In this regard, the software provides effective interoperability. The new standard has been tested by experiments and data exchange between Nanyang Technological University in Singapore and Imperial College London.

  • Journal article
    Schuster C, Bellows L, Tosi T, Campeotto, Corrigan, Freemont P, Grundling Aet al., 2016,

    The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus

    , Science Signaling, Vol: 9, Pages: ra81-ra81, ISSN: 1945-0877

    Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that the LAC*ΔgdpP strain of S. aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

  • Journal article
    Galizi R, Hammond A, Kyrou K, Taxiarchi C, Bernardini F, O'Loughlin SM, Papathanos PA, Nolan T, Windbichler N, Crisanti Aet al., 2016,

    A CRISPR-Cas9 sex-ratio distortion system for genetic control.

    , Scientific Reports, Vol: 6, ISSN: 2045-2322

    Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.

  • Journal article
    Borkowski O, Ceroni F, Stan GB, Ellis Tet al., 2016,

    Overloaded and stressed: whole-cell considerations for bacterial synthetic biology.

    , Current Opinion in Microbiology, Vol: 33, Pages: 123-130, ISSN: 1879-0364

    The predictability and robustness of engineered bacteria depend on the many interactions between synthetic constructs and their host cells. Expression from synthetic constructs is an unnatural load for the host that typically reduces growth, triggers stresses and leads to decrease in performance or failure of engineered cells. Work in systems and synthetic biology has now begun to address this through new tools, methods and strategies that characterise and exploit host-construct interactions in bacteria. Focusing on work in E. coli, we review here a selection of the recent developments in this area, highlighting the emerging issues and describing the new solutions that are now making the synthetic biology community consider the cell just as much as they consider the construct.

  • Journal article
    Borkowski O, Gilbert C, Ellis T, 2016,

    SYNTHETIC BIOLOGY. On the record with E. coli DNA.

    , Science, Vol: 353, Pages: 444-445, ISSN: 0036-8075
  • Journal article
    Liu Z, Yoshihara A, Kelly C, Heap JT, Marqvorsen MH, Jenkinson SF, Wormald MR, Otero JM, Estévez A, Kato A, Fleet GW, Estévez RJ, Izumori Ket al., 2016,

    6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology

    , Chemistry, Vol: 22, Pages: 12557-12565, ISSN: 1521-3765

    In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

  • Journal article
    Klymenko O, Royle K, Polizzi KM, Shah N, Kontoravdi Cet al., 2016,

    Designing an Artificial Golgi Reactor to achieve targeted glycosylation of monoclonal antibodies

    , AICHE Journal, Vol: 62, Pages: 2959-2973, ISSN: 0001-1541

    The therapeutic efficacy of monoclonal antibodies (mAbs) is dependent upon their glycosylationpatterns. As the largest group of currently approved biopharmaceuticals, the microheterogeneity inmAb oligosaccharide profiles deriving from mammalian cell production is a challenge to thebiopharmaceutical industry. Disengaging the glycosylation process from the cell may offer significantenhancement of product quality and allow better control and reproducibility in line with the Quality byDesign paradigm. Three potential designs of an Artificial Golgi reactor implementing targeted sequentialglycosylation of mAbs are proposed including a (i) microcapillary film reactor, (ii) packed bed reactorwith non-porous pellets, and (iii) packed bed reactor with porous pellets. Detailed mathematical modelsare developed to predict their performance for a range of design and operational parameters. While allthree reactor designs can achieve desired conversion levels, the choice of a particular one depends onthe required throughput and the associated cost of enzymes and co-substrates.

  • Journal article
    Jimenez del Val I, Polizzi K, Kontoravdi C, 2016,

    A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

    , Scientific Reports, Vol: 6, ISSN: 2045-2322

    Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation.

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