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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
    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.

  • Conference paper
    Pan W, Yuan Y, Ljung L, Gonçalves JM, Stan G-Bet al., 2016,

    Identifying biochemical reaction networks from heterogeneous datasets

    , 2015 IEEE 54th Annual Conference on Decision and Control (CDC), Publisher: IEEE, Pages: 2525-2530

    In this paper, we propose a new method to identify biochemical reaction networks (i.e. both reactions and kinetic parameters) from heterogeneous datasets. Such datasets can contain (a) data from several replicates of an experiment performed on a biological system; (b) data measured from a biochemical network subjected to different experimental conditions, for example, changes/perturbations in biological inductions, temperature, gene knock-out, gene over-expression, etc. Simultaneous integration of various datasets to perform system identification has the potential to avoid non-identifiability issues typically arising when only single datasets are used.

  • Journal article
    Aw R, Polizzi KM, 2016,

    Liquid PTVA: A faster and cheaper alternative for generating multi-copy clones in Pichia pastoris

    , Microbial Cell Factories, Vol: 15, ISSN: 1475-2859

    BACKGROUND:Multiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming.RESULTS:We have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes.CONCLUSIONS:We present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.

  • Journal article
    Sootla A, Oyarzun DA, Angeli D, Stan GBet al., 2016,

    Shaping Pulses to Control Bistable Systems: Analysis, Computation and Counterexamples

    , Automatica, Vol: 63, Pages: 254-264, ISSN: 1873-2836

    In this paper we study how to shape temporal pulses to switch a bistable system between its stable steady states. Our motivation forpulse-based control comes from applications in synthetic biology, where it is generally difficult to implement real-time feedback controlsystems due to technical limitations in sensors and actuators. We show that for monotone bistable systems, the estimation of the set ofall pulses that switch the system reduces to the computation of one non-increasing curve. We provide an efficient algorithm to computethis curve and illustrate the results with a genetic bistable system commonly used in synthetic biology. We also extend these results tomodels with parametric uncertainty and provide a number of examples and counterexamples that demonstrate the power and limitationsof the current theory. In order to show the full potential of the framework, we consider the problem of inducing oscillations in a monotonebiochemical system using a combination of temporal pulses and event-based control. Our results provide an insight into the dynamics ofbistable systems under external inputs and open up numerous directions for future investigation.

  • Conference paper
    Reeve AB, Petkiewicz S, Hagemann H, Santosa G, Florea M, Ellis Tet al., 2016,

    Modified bacterial nanocellulose as a bioadsorbent material

  • Conference paper
    Kelwick R, Webb AJ, Macdonald JT, Freemont PSet al., 2016,

    Development of a bacillus subtilis cell-free transcriptiontranslation system

  • Conference paper
    Kopniczky M, Jensen K, Freemont P, 2016,

    Introducing the human cell-free TX-TL system as a new prototyping platform for mammalian synthetic biology

  • Conference paper
    Polizzi KM, Freemont PS, 2016,

    Synthetic biology biosensors for healthcare and industrial biotechnology applications

  • Journal article
    Pan W, Yuan Y, Goncalves J, Stan G-Bet al., 2015,

    A Sparse Bayesian Approach to the Identification of Nonlinear State-Space Systems

    , IEEE Transactions on Automatic Control, Vol: 61, Pages: 182-187, ISSN: 1558-2523

    This technical note considers the identification ofnonlinear discrete-time systems with additive process noise butwithout measurement noise. In particular, we propose a methodand its associated algorithm to identify the system nonlinear functionalforms and their associated parameters from a limited numberof time-series data points. For this, we cast this identificationproblem as a sparse linear regression problem and take a Bayesianviewpoint to solve it. As such, this approach typically leads tononconvex optimizations. We propose a convexification procedurerelying on an efficient iterative re-weighted 1-minimization algorithmthat uses general sparsity inducing priors on the parametersof the system and marginal likelihood maximisation. Using thisapproach, we also show how convex constraints on the parameterscan be easily added to the proposed iterative re-weighted1-minimization algorithm. In the supplementary material availableonline (arXiv:1408.3549), we illustrate the effectiveness of theproposed identification method on two classical systems in biologyand physics, namely, a genetic repressilator network and a largescale network of interconnected Kuramoto oscillators.

  • Journal article
    Oyarzun DA, Chaves M, 2015,

    Design of a bistable switch to control cellular uptake

    , Journal of the Royal Society Interface, Vol: 20150618, ISSN: 1742-5689

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