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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 articleDebalke S, Habtewold T, Duchateau L, et al., 2019,
The effect of silencing immunity related genes on longevity in a naturally occurring Anopheles arabiensis mosquito population from southwest Ethiopia
, Parasites & Vectors, Vol: 12, ISSN: 1756-3305BackgroundVector control remains the most important tool to prevent malaria transmission. However, it is now severely constrained by the appearance of physiological and behavioral insecticide resistance. Therefore, the development of new vector control tools is warranted. Such tools could include immunization of blood hosts of vector mosquitoes with mosquito proteins involved in midgut homeostasis (anti-mosquito vaccines) or genetic engineering of mosquitoes that can drive population-wide knockout of genes producing such proteins to reduce mosquito lifespan and malaria transmission probability.MethodsTo achieve this, candidate genes related to midgut homeostasis regulation need to be assessed for their effect on mosquito survival. Here, different such candidate genes were silenced through dsRNA injection in the naturally occurring Anopheles arabiensis mosquitoes and the effect on mosquito survival was evaluated.ResultsSignificantly higher mortality rates were observed in the mosquitoes silenced for FN3D1 (AARA003032), FN3D3 (AARA007751) and GPRGr9 (AARA003963) genes as compared to the control group injected with dsRNA against a non-related bacterial gene (LacZ). This observed difference in mortality rate between the candidate genes and the control disappeared when gene-silenced mosquitoes were treated with antibiotic mixtures, suggesting that gut microbiota play a key role in the observed reduction of mosquito survival.ConclusionsWe demonstrated that interference with the expression of the FN3D1, FN3D3 or GPRGr9 genes causes a significant reduction of the longevity of An. arabiensis mosquito in the wild.
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Journal articleGoey CH, Bell D, Kontoravdi C, 2019,
CHO cell cultures in shake flasks and bioreactors present different host cell protein profiles in the supernatant
, Biochemical Engineering Journal, Vol: 144, Pages: 185-192, ISSN: 1369-703XSeveral studies on the impact of cell culture parameters on the profile of host cell protein (HCP) impurities have been carried out in shake flasks. Herein, we explore how transferable the findings and conclusions of such investigations are to lab-scale bioreactors. Experiments were performed in both systems in fed-batch mode under physiological temperature and with a shift to mild hypothermia and the impact on key upstream performance indicators was quantified. Under both temperatures, bioreactors produced a richer HCP pool despite the overall concentration being similar at both scales and temperatures. The number of different HCPs detected in bioreactor supernatants was four times higher than that in flasks under physiological temperature and more than eight times higher under mild hypothermia. The origin of HCPs was also altered from mostly naturally secreted proteins in flasks to mainly intracellular proteins in bioreactors at the lower temperature. Although the number of species correlated with apoptotic cell density in bioreactors, this was not the case in flasks. Even though the level of HCP impurities and mAb/HCP concentration ratio were similar under all four conditions with an average of approximately 330 μg HCP/mL culture and 0.3 mg HCP/mg IgG4, respectively, the fact that culture method significantly affects the number of species present in the supernatant can have implications for downstream processing steps.
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Journal articleStopnitzky E, Still S, Ouldridge TE, et al., 2019,
Physical limitations of work extraction from temporal correlations.
, Physical Review E, Vol: 99, ISSN: 1539-3755Recently proposed information-exploiting systems extract work from a single heat bath by using temporal correlations on an input tape. We study how enforcing time-continuous dynamics, which is necessary to ensure that the device is physically realizable, constrains possible designs and drastically diminishes efficiency. We show that these problems can be circumvented by means of applying an external, time-varying protocol, which turns the device from a "passive," free-running machine into an "actively" driven one.
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Journal articleRuiz VMR, Sousa GL, Sneed SD, et al., 2019,
Stimulation of a protease targeting the LRIM1/APL1C complex reveals specificity in complement-like pathway activation in Anopheles gambiae
, PLoS One, Vol: 14, Pages: 1-14, ISSN: 1932-6203The complement-like pathway of the African malaria mosquito Anopheles gambiae provides protection against infection by diverse pathogens. A functional requirement for a core set of proteins during infections by rodent and human malaria parasites, bacteria, and fungi suggests a similar mechanism operates against different pathogens. However, the extent to which the molecular mechanisms are conserved is unknown. In this study we probed the biochemical responses of complement-like pathway to challenge by the Gram-positive bacterium Staphyloccocus aureus. Western blot analysis of the hemolymph revealed that S. aureus challenge activates a TEP1 convertase-like activity and promotes the depletion of the protein SPCLIP1. S. aureus challenge did not lead to an apparent change in the abundance of the LRIM1/APL1C complex compared to challenge by the Gram-negative bacterium, Escherichia coli. Following up on this observation using a panel of LRIM1 and APL1C antibodies, we found that E. coli challenge, but not S. aureus, specifically activates a protease that cleaves the C-terminus of APL1C. Inhibitor studies in vivo and in vitro protease assays suggest that a serine protease is responsible for APL1C cleavage. This study reveals that despite different challenges converging on activation of a TEP1 convertase-like activity, the mosquito complement-like pathway also includes pathogen-specific reactions.
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Journal articleMcCarty NS, Shaw WM, Ellis T, et al., 2019,
Rapid assembly of gRNA arrays via modular cloning in yeast
, ACS Synthetic Biology, Vol: 8, Pages: 906-910, ISSN: 2161-5063CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.
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Journal articleBoo A, Ellis T, Stan G, 2019,
Host-aware synthetic biology
, Current Opinion in Systems Biology, Vol: 14, Pages: 66-72, ISSN: 2452-3100Unnatural gene expression imposes a load on engineered microorganisms thatdecreases their growth and subsequent production yields, a phenomenon knownasburden. In the last decade, the field of synthetic biology has made progress onthe development of biomolecular feedback control systems and other approachesthat can improve the growth of engineered cells, as well as the genetic stability,portability and robust performance of cell-hosted synthetic constructs. In thisreview, we highlight recent work focused on the development of host-aware syn-thetic biology.
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Journal articleKotidis P, Kontoravdi C, 2019,
Strategic framework for parameterization of cell culture models
, Processes, Vol: 7, ISSN: 2227-9717Global Sensitivity Analysis (GSA) is a technique that numerically evaluates the significance of model parameters with the aim of reducing the number of parameters that need to be estimated accurately from experimental data. In the work presented herein, we explore different methods and criteria in the sensitivity analysis of a recently developed mathematical model to describe Chinese hamster ovary (CHO) cell metabolism in order to establish a strategic, transferable framework for parameterizing mechanistic cell culture models. For that reason, several types of GSA employing different sampling methods (Sobol’, Pseudo-random and Scrambled-Sobol’), parameter deviations (10%, 30% and 50%) and sensitivity index significance thresholds (0.05, 0.1 and 0.2) were examined. The results were evaluated according to the goodness of fit between the simulation results and experimental data from fed-batch CHO cell cultures. Then, the predictive capability of the model was tested against four different feeding experiments. Parameter value deviation levels proved not to have a significant effect on the results of the sensitivity analysis, while the Sobol’ and Scrambled-Sobol’ sampling methods and a 0.1 significance threshold were found to be the optimum settings. The resulting framework was finally used to calibrate the model for another CHO cell line, resulting in a good overall fit. The results of this work set the basis for the use of a single mechanistic metabolic model that can be easily adapted through the proposed sensitivity analysis method to the behavior of different cell lines and therefore minimize the experimental cost of model development.
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Journal articleWen Z, Ledesma-Amaro R, Lin J, et al., 2019,
Improved n-butanol production from Clostridium cellulovorans by integrated metabolic and evolutionary engineering
, Applied and Environmental Microbiology, Vol: 85, ISSN: 0099-2240Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a CoA-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing genes of adhE1 and ctfA-ctfB-adc from C. acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans, to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans enhanced 138-fold from less than 0.025 g/L to 3.47 g/L. This method represents a milestone toward n-butanol production by CBP, using single recombinant clostridia. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.
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Journal articleKuntz J, Thomas P, Stan G-B, et al., 2019,
The exit time finite state projection scheme: bounding exit distributions and occupation measures of continuous-time Markov chains
, SIAM Journal on Scientific Computing, Vol: 41, Pages: A748-A769, ISSN: 1064-8275We introduce the exit time finite state projection (ETFSP) scheme, a truncation- based method that yields approximations to the exit distribution and occupation measure associated with the time of exit from a domain (i.e., the time of first passage to the complement of the domain) of time-homogeneous continuous-time Markov chains. We prove that: (i) the computed approximations bound the measures from below; (ii) the total variation distances between the approximations and the measures decrease monotonically as states are added to the truncation; and (iii) the scheme converges, in the sense that, as the truncation tends to the entire state space, the total variation distances tend to zero. Furthermore, we give a computable bound on the total variation distance between the exit distribution and its approximation, and we delineate the cases in which the bound is sharp. We also revisit the related finite state projection scheme and give a comprehensive account of its theoretical properties. We demonstrate the use of the ETFSP scheme by applying it to two biological examples: the computation of the first passage time associated with the expression of a gene, and the fixation times of competing species subject to demographic noise.
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Journal articleFriddin MS, Elani Y, Trantidou T, et al., 2019,
New directions for artificial cells using rapid prototyped biosystems
, Analytical Chemistry, Vol: 91, Pages: 4921-4928, ISSN: 0003-2700Microfluidics has been shown to be capable of generating a range of single- and multi- compartment vesicles and bilayer delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly recognized as powerful biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems and for screening drugs. One bottleneck in developing this technology is the time, expertise and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from Computer Aided Design (CAD) drawings. We highlight how this rapid prototyping revolution is transforming the fabrication of microfluidic devices for bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multi-billion dollar start-up could emerge from an attic or a basement.
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Journal articleStan G, Ellis T, Boo A, 2019,
Host-Aware Synthetic Biology
, Current Opinion in Systems Biology, ISSN: 2452-3100 -
Journal articleChristophides G, Rona L, Cechetto Carlos B, et al., 2019,
A comprehensive analysis of malaria transmission in Brazil
, Pathogens and Global Health, Vol: 113, Pages: 1-13, ISSN: 2047-7724Malaria remains a serious public health problem in Brazil despite a significant drop in the number of cases in the past decade. We conduct a comprehensive analysis of malaria transmission in Brazil to highlight the epidemiologically most relevant components that could help tackle the disease. We consider factors impacting on the malaria burden and transmission dynamics including the geographical occurrence of both autochthonous and imported infections, the distribution and abundance of malaria vectors and records of natural mosquito infections with Plasmodium. Our analysis identifies three discrete malaria transmission systems related to the Amazon rainforest, Atlantic rainforest and Brazilian coast, respectively. The Amazonian system accounts for 99% of all malaria cases in the country. It is largely due to autochthonous P. vivax and P. falciparum transmission by mosquitoes of the Nyssorhynchus subgenus, primarily Anopheles darlingi. Whilst P. vivax transmission is widespread, P. falciparum transmission is restricted to hotspot areas mostly in the States of Amazonas and Acre. This system is the major source of P. vivax exportation to the extra-Amazonian regions that are also affected by importation of P. falciparum from Africa. The Atlantic system comprises autochthonous P. vivax transmission typically by the bromeliad-associated mosquitoes An. cruzii and An. bellator of the Kerteszia subgenus. An. cruzii also transmits simian malaria parasites to humans. The third, widespread but geographically fragmented, system is found along the Brazilian coast and comprises P. vivax transmission mainly by An. aquasalis. We conclude that these geographically and biologically distinct malaria transmission systems require specific strategies for effective disease control.
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Conference paperThaore V, Moore S, Polizzi K, et al., 2019,
Cell-free multi-enzyme system for the industrial production of fine chemicals
, Chemical Engineering Day UK 2019 -
Journal articleCes O, Elani Y, 2019,
Community building in synthetic biology.
, Experimental biology and medicine (Maywood, N.J.), Vol: 244, Pages: 281-282, ISSN: 0037-9727 -
Journal articleCelinska E, Borkowska M, Bialas W, et al., 2019,
Genetic engineering of Ehrlich pathway modulate production of higher alcohols in engineered Yarrowia lipolytica
, FEMS Yeast Research, Vol: 19, ISSN: 1567-1356Microbial cells can produce a vast spectrum of chemical compounds, including those most desired by the global chemical market, as for example higher alcohols, which are promising alternative fuels and chemical feedstock. In the current research, we investigated the effects of the Ehrlich pathway genetic engineering on higher alcohols production in Y. lipolytica, which directly follows our previous findings concerning elucidation of putative molecular identities involved in this pathway. To this end, we constructed two alternative expression cassettes composed of previously identified genes, putatively involved in the Ehrlich pathway in Y. lipolytica, and cloned them under the control of constitutive pTEF promoter, and by this-released them from extensive native regulation. The effects of the pathway engineering were investigated upon provision of different, Ehrlich pathway-inducing amino acids (L-Phe, L-Leu, L-Ile and L-Val). In general, amplification of the Ehrlich pathway in many cases led to increased formation of a respective higher alcohol from its precursor. We observed interesting effects of aminotransferase BAT2 deletion on synthesis of 2-phenylethanol and its acetate ester, significant relationship between L-Val and L-Phe catabolic pathways, and extensive 'cross-induction' of the derivative compounds synthesis by non-direct precursors.
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Journal articleMcAleer MA, Jakasa I, Hurault G, et al., 2019,
Systemic and stratum corneum biomarkers of severity in infant AD include markers of innate and Th-related immunity and angiogenesis
, British Journal of Dermatology, Vol: 180, Pages: 586-596, ISSN: 1365-2133BACKGROUND: Biomarkers of atopic dermatitis (AD) are largely lacking, especially in infant AD. Those that have been examined to date have focused mostly on serum cytokines with few on non-invasive biomarkers in the skin. OBJECTIVES: We aimed to explore biomarkers obtainable from non-invasive sampling of infant skin. We compared these to plasma biomarkers and structural and functional measures of the skin barrier. METHODS: We recruited 100 infants at first presentation with AD, who were treatment naïve to topical or systemic anti-inflammatory therapies and 20 healthy children. We sampled clinically unaffected skin by tape stripping the stratum corneum (SC). Multiple cytokines and chemokines and natural moisturizing factors (NMF) were measured in the SC and plasma. We recorded disease severity and skin barrier function. RESULTS: 19 SC and 12 plasma biomarkers showed significant difference between healthy and AD skin. Some biomarkers were common to both the SC and plasma, and others were compartment-specific. Identified biomarkers of AD severity included Th2 skewed markers (IL-13, CCL17, CCL22, IL-5), markers of innate activation (IL-18, Il-1α, IL1β, CXCL8), angiogenesis (Flt-1, VEGF) and others (sICAM-1, vCAM-1, IL-16, IL-17A). CONCLUSIONS: We identified clinically relevant biomarkers of AD, including novel markers, easily sampled and typed in infants. These markers may provide objective assessment of disease severity and suggest new therapeutic targets, or response measurement targets for AD. Future studies will be required to determine if these biomarkers, seen in very early AD, can predict disease outcomes or comorbidities.
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Journal articleAw R, Polizzi KM, 2019,
Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform
, Biotechnology and Bioengineering, Vol: 116, Pages: 656-666, ISSN: 0006-3592Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a 3‐fold increase in recombinant protein yield compared to those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg mL‐1. Therefore, this work not only adds to the growing number of CFPS systems from diverse organisms, but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.
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Journal articleSuckling L, McFarlane C, Sawyer C, et al., 2019,
Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation
, Synthetic and Systems Biotechnology, Vol: 4, Pages: 57-66, ISSN: 2405-805XHigh-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.
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Journal articleJamshidiha M, Pérez-Dorado I, Murray JW, et al., 2019,
Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a
, Acta Crystallographica Section D Structural Biology, Vol: 75, Pages: 342-353, ISSN: 2059-7983Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27a<jats:sup>Mut</jats:sup>, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by <jats:italic>Phaser</jats:italic> were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in <jats:italic>Phaser</jats:italic>. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in <jats:italic>Phaser</jats:italic>, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more sui
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Journal articleKylilis N, Riangrungroj P, Lai H-E, et al., 2019,
Whole-cell biosensor with tuneable limit of detection enables low-cost agglutination assays for medical diagnostic applications
, ACS Sensors, Vol: 4, Pages: 370-378, ISSN: 2379-3694Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date, the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design can detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straight-forward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanbodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.
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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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