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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 articleMacDonald JT, Kabasakal BV, Godding D, et 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: 1091-6490The 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.
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Journal articleAgustín-Pavón C, Mielcarek M, Garriga-Canut M, et 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: 1750-1326Background: 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
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Journal articleToczek M, Zielonka D, Zukowska P, et 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: 0925-4439Huntington'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.
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Journal articleCeroni F, Blount BA, Ellis T, 2016,
Sensing the Right Time to Be Productive
, Cell Systems, Vol: 3, Pages: 116-117, ISSN: 2405-4720Engineered E. coli can be made to autonomously switch from growth to production by a modular two-gate system that reduces the burden of biosynthesis.
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Journal articleSainz 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: 2161-5063This 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.
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Journal articleSchuster C, Bellows L, Tosi T, et 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: 1945-0877Staphylococcus 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.
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Journal articleGalizi R, Hammond A, Kyrou K, et al., 2016,
A CRISPR-Cas9 sex-ratio distortion system for genetic control.
, Scientific Reports, Vol: 6, ISSN: 2045-2322Genetic 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.
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Journal articleBorkowski O, Ceroni F, Stan GB, et al., 2016,
Overloaded and stressed: whole-cell considerations for bacterial synthetic biology.
, Current Opinion in Microbiology, Vol: 33, Pages: 123-130, ISSN: 1879-0364The 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.
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Journal articleBorkowski 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 articleLiu Z, Yoshihara A, Kelly C, et 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: 1521-3765In 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.
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Journal articleKlymenko O, Royle K, Polizzi KM, et al., 2016,
Designing an Artificial Golgi Reactor to achieve targeted glycosylation of monoclonal antibodies
, AICHE Journal, Vol: 62, Pages: 2959-2973, ISSN: 0001-1541The 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.
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Journal articleJimenez 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: 2045-2322Glycosylation 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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Journal articleFilloux A, Freemont P, 2016,
Structural biology: baseplates in contractile machines
, Nature Microbiology, Vol: 1, ISSN: 2058-5276 -
Journal articleReeve B, Martinez Klimova E, de Jonghe J, et al., 2016,
The Geobacillus plasmid set: a modular toolkit for thermophile engineering
, ACS Synthetic Biology, Vol: 5, Pages: 1342-1347, ISSN: 2161-5063Geobacillus 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.
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Journal articleChambers 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: 1470-8752The 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.
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Journal articleFlorea M, Hagemann H, Santosa G, et 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: 0027-8424Bacterial 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.
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Journal articlePlanamente S, Salih O, Manoli E, et al., 2016,
TssA forms a gp6-like ring attached to the type VI secretion sheath
, EMBO Journal, Vol: 35, Pages: 1613-1627, ISSN: 0261-4189The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo. In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS. Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.
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Journal articleBlount BA, Driessen MRM, Ellis T, 2016,
GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA
, Scientific Reports, Vol: 6, ISSN: 2045-2322Existing 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.
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Journal articleLund-Palau H, Turnbull AR, Bush A, et al., 2016,
Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches
, Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.
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Journal articleWebb AJ, Kelwick R, Doenhoff MJ, et al., 2016,
A protease-based biosensor for the detection of schistosome cercariae
, Scientific Reports, Vol: 6, ISSN: 2045-2322Parasitic 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.
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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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