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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
    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
    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, 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
    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
    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
    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.

  • Journal article
    Reeve B, Martinez Klimova E, de Jonghe J, Leak DJ, Ellis Tet al., 2016,

    The Geobacillus plasmid set: a modular toolkit for thermophile engineering

    , ACS Synthetic Biology, Vol: 5, Pages: 1342-1347, ISSN: 2161-5063

    Geobacillus thermoglucosidasius is agram-positive thermophile of industrial interest thatexhibits rapid growth and can utilize a variety ofplant-derived feedstocks. It is an attractive chassisorganism for high temperature biotechnology andsynthetic biology applications but is currently limitedby a lack of available genetic tools. Here we describea set of modular shuttle vectors, including apromoter library and reporter proteins. The compactplasmids are composed of interchangeable modulesfor molecular cloning in Escherichia coli and stablepropagation in G. thermoglucosidasius and otherGeobacillus species. Modules include two origins ofreplication, two selectable markers and three reporterproteins for characterization of gene expression.For fine-tuning heterologous expression from theseplasmids, we include a characterized promoter libraryand test ribosome binding site design. Together,these gene expression tools and a standardizedplasmid set can facilitate modularity and part exchangeto make Geobacillus a thermophile chassis forsynthetic biology.

  • Journal article
    Chambers S, Kitney R, Freemont P, 2016,

    The Foundry: the DNA synthesis and construction Foundry at Imperial College.

    , Biochemical Society Transactions, Vol: 44, Pages: 687-688, ISSN: 1470-8752

    The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms.

  • Journal article
    Blount BA, Driessen MRM, Ellis T, 2016,

    GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA

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

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb.

  • Journal article
    Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont P, Kitney RI, Reeve B, Ellis Tet al., 2016,

    Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E3431-E3440, ISSN: 1091-6490

    Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity and biocompatibility make it of great interest to materials science, however precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a new strain of Komagataeibacter rhaeticus (Komagataeibacter rhaeticus iGEM) that can produce cellulose at high yields, grow in low nitrogen conditions, and is highly resistant to toxic chemicals. We achieve external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering and biotechnology.

  • Journal article
    Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald J, Wen KY, Mckeown C, Baldwin G, Ellis T, Jensen K, Freemont PSet al., 2016,

    A protease-based biosensor for the detection of schistosome cercariae

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

    Parasitic diseases affect millions of people worldwide, causing debilitating illnesses anddeath. Rapid and cost-effective approaches to detect parasites are needed, especially inresource-limited settings. A common signature of parasitic diseases is the release of specificproteases by the parasites at multiple stages during their life cycles. To this end, weengineered several modular Escherichia coli and Bacillus subtilis whole-cell-basedbiosensors which incorporate an interchangeable protease recognition motif into theirdesigns. Herein, we describe how several of our engineered biosensors have been applied todetect the presence and activity of elastase, an enzyme released by the cercarial larvae stageof Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes areresponsible for the infection of an estimated 200 million people worldwide. Since ourbiosensors are maintained in lyophilised cells, they could be applied for the detection of S.mansoni and other parasites in settings without reliable cold chain access.

  • Journal article
    Awan AR, Shaw WM, Ellis T, 2016,

    Biosynthesis of therapeutic natural products using synthetic biology

    , Advanced Drug Delivery Reviews, Vol: 105, Pages: 96-106, ISSN: 1872-8294

    Natural products are a group of bioactive structurally diverse chemicals produced by microorganisms and plants. These molecules and their derivatives have contributed to over a third of the therapeutic drugs produced in the last century. However, over the last few decades traditional drug discovery pipelines from natural products have become far less productive and far more expensive. One recent development with promise to combat this trend is the application of synthetic biology to therapeutic natural product biosynthesis. Synthetic biology is a young discipline with roots in systems biology, genetic engineering, and metabolic engineering. In this review, we discuss the use of synthetic biology to engineer improved yields of existing therapeutic natural products. We further describe the use of synthetic biology to combine and express natural product biosynthetic genes in unprecedented ways, and how this holds promise for opening up completely new avenues for drug discovery and production.

  • Journal article
    Florea M, Reeve B, Abbott J, Freemont PS, Ellis Tet al., 2016,

    Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

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

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

  • Journal article
    Yu N, Nützmann HW, MacDonald JT, Moore B, Field B, Berriri S, Trick M, Rosser SJ, Kumar SV, Freemont PS, Osbourn Aet al., 2016,

    Delineation of metabolic gene clusters in plant genomes by chromatin signatures.

    , Nucleic Acids Research, Vol: 44, Pages: 2255-2265, ISSN: 1362-4962

    Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.

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