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 articlede Martín Garrido N, Crone MA, Ramlaul K, et al., 2020,
Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
Journal articleWen Z, Lu M, Ledesma-Amaro R, et al., 2020,
Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delayed development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which made it very suitable for gene disruption of Clostridium. The application of TargeTron technology in Clostridium was academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time-saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone-butanol-ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis & reconstruction. In the review, we revisited 12 years' advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application and efforts to expand its capabilities, or to avoid potential drawbacks. Some other technologies as putative competitors or collaborators are also discussed. We believe that TargeTron combined with CRISPR/Cas-assisted gene/base editing and gene-expression regulation system will make a better future for clostridial genetic modification.
Book chapterSootla A, Stan G-B, Ernst D, 2020,
© Springer Nature Switzerland AG 2020. Koopman operator theory offers numerous techniques for analysis and control of complex systems. In particular, in this chapter we will argue that for the problem of convergence to an equilibrium, the Koopman operator can be used to take advantage of the geometric properties of controlled systems, thus making the optimal solutions more transparent, and easier to analyze and implement. The motivation for the study of the convergence problem comes from biological applications, where easy-to-implement and easy-to-analyze solutions are of particular value. At the moment, theoretical results have been developed for a class of nonlinear systems called monotone systems. However, the core ideas presented here can be applied heuristically to non-monotone systems. Furthermore, the convergence problem can serve as a building block for solving other control problems such as switching between stable equilibria or inducing oscillations. These applications are illustrated in biologically inspired numerical examples.
Journal articleStorch M, Dwijayanti A, Mallick H, et al., 2020,
Journal articleAngrisano F, Sala K, Tapanelli S, et al., 2019,
Male-specific protein disulphide isomerase function is essential for plasmodium transmission and a vulnerable target for intervention, Scientific Reports, Vol: 9, ISSN: 2045-2322
Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.
Journal articleJezierska S, Claus S, Ledesma-Amaro R, et al., 2019,
Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production, Journal of Industrial Microbiology and Biotechnology, Vol: 46, Pages: 1697-1706, ISSN: 0169-4146
Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300 g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and β-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933 g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.
Journal articleScholes NS, Schnoerr D, Isalan M, et al., 2019,
Journal articleCasula E, Traversari G, Fadda S, et al., 2019,
Journal articleGowers G-OF, Cameron SJS, Perdones-Montero A, et al., 2019,
Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) - a form of ambient laser desorption ionisation mass spectrometry - to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies in under 4 hours, while simultaneously generating recoverable glycerol stocks of each colony in real-time. This showcases LA-REIMS as a pre-screening tool to complement downstream quantification methods such as LCMS. Through pre-screening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.
Conference paperWebb AJ, Kelwick R, Wang Y, et al., 2019,
AL-PHA beads: bioplastic-bsaed protease biosensors for global health, British Society for Parasitology Autumn Symposium, Belfast, UK
Journal articleFasulo B, Meccariello A, Morgan M, et al., 2019,
<jats:p>Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established<jats:italic>Drosophila</jats:italic>models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - “X-shredding” and “X-meddling” - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.</jats:p>
Journal articleDahalan FA, Churcher TS, Windbichler N, et al., 2019,
The male mosquito contribution towards malaria transmission: Mating influences the Anopheles female midgut transcriptome and increases female susceptibility to human malaria parasites., PLoS Pathogens, Vol: 15, Pages: 1-19, ISSN: 1553-7366
Mating causes dramatic changes in female physiology, behaviour, and immunity in many insects, inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20-hydroxyecdysone. This hormone, which is also induced by blood-feeding, plays a major role in activating vitellogenesis for egg production. Here we show that female Anopheles coluzzii susceptibility to Plasmodium falciparum infection is significantly higher in mated females compared to virgins. We also find that mating status has a major impact on the midgut transcriptome, detectable only under sugar-fed conditions: once females have blood-fed, the transcriptional changes that are induced by mating are likely masked by the widespread effects of blood-feeding on gene expression. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, we mimicked mating by injecting virgin females with 20E, finding that these females are significantly more susceptible to human malaria parasites than virgin females injected with the control 20E carrier. Further RNAseq was carried out to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. We find that 79 midgut-expressed genes are regulated in common by both mating and 20E, and 96% (n = 76) of these are regulated in the same direction (up vs down in 20E/mated). Together, these findings show that male Anopheles mosquitoes induce changes in the female midgut that can affect female susceptibility to P. falciparum. This implies that in nature, males might contribute to malaria transmission in previously unappreciated ways, and that vector control strategies that target males may have additional benefits towards reducing transm
Journal articleGowers G-O, Vince O, Charles J-H, et al., 2019,
Entirely off-grid and solar-powered DNA sequencing of microbial communities during an ice cap traverse expedition, Genes, Vol: 10, Pages: 1-10, ISSN: 2073-4425
Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth’s surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe’s largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition.
Journal articleLai H-E, Canavan C, Cameron L, et al., 2019,
Synthetic biology is a rapidly emerging interdisciplinary field of science and engineering that aims to redesign living systems through reprogramming genetic information. The field has catalysed global debate among policymakers and publics. Here we describe how synthetic biology relates to these international deliberations, particularly the Convention on Biological Diversity (CBD).
Journal articleJiménez A, Muñoz-Fernández G, Ledesma-Amaro R, et al., 2019,
The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner.
Journal articleGiese B, Friess JL, Barton NH, et al., 2019,
Journal articleOstrov N, Beal J, Ellis T, et al., 2019,
Engineering biology with recombinant DNA, broadly called synthetic biology, has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of molecular tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biology because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnology.
Journal articleMcFarlane C, Shah N, Kabasakal B, et al., 2019,
Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) are essential Calvin-Benson cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyses the reduction step of the CB cycle with NADPH to produce the sugar, glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are co-regulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of Calvin-Benson cycle regulation by CP12 is unknown. Here we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologues, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.
Journal articleFreemont P, 2019,
Synthetic biology is a rapidly emerging interdisciplinary research field that is primarily built upon foundational advances in molecular biology combined with engineering design. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies. This has spurned a rapid growth in start-up companies and the new synthetic biology industry is growing rapidly, with start-up companies receiving ~$6.1B investment since 2015 and a global synthetic biology market value estimated to be $14B by 2026. Many of the new start-upscan be grouped within a multi-layer ‘technology stack’. The ‘stack’ comprises a number of technology layers which together can be applied to a diversity of new biotechnology applications like consumer biotechnology products and living therapies. The ‘stack’ also enables new commercial opportunities and value chains similar to the software design and manufacturing revolution of the 20th century. However, synthetic biology industry is at a crucial point, as it now requires recognisable commercial successes in order for the industry to expand and scale, in terms of investment and companies. However, such expansion may directly challenge the ethos of synthetic biology, in terms of open technology sharing and democratisation, which could by accident lead to multi-national corporations and technology monopolies similar to the existing biotechnology/biopharma industry.
Journal articleWood TE, Howard SA, Forster A, et al., 2019,
The Pseudomonas aeruginosa T6SS delivers a periplasmic toxin that disrupts bacterial cell morphology, Cell Reports, Vol: 29, Pages: 187-201.e7, ISSN: 2211-1247
The type VI secretion system (T6SS) is crucialin interbacterial competition and is avirulence determinant ofmany Gram-negative bacteria. Several T6SS effectorsarecovalently fused to secreted T6SS structural components such asthe VgrG spike for delivery into target cells.In Pseudomonas aeruginosa, theVgrG2b effector waspreviously proposedto mediatebacterial internalisation into eukaryotic cells. In this work, wefind that the VgrG2b C-terminal domain(VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein.We resolve thestructure of VgrG2bC-ter and confirm it is a member ofthezinc-metallopeptidasefamily of enzymes. We show that this effector causesmembrane blebbing atmidcell, whichsuggests a distincttype of T6SS-mediated growthinhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Ourstudyintroduces a further effector family to the T6SS arsenaland demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.
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