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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
    Det-Udom R, Gilbert C, Liu L, Prakitchaiwattana C, Ellis T, Ledesma Amaro Ret al., 2019,

    Towards semi-synthetic microbial communities: Enhancing soy sauce fermentation properties in B. subtilis co-cultures

    , Microbial Cell Factories, Vol: 18, ISSN: 1475-2859

    BackgroundMany fermented foods and beverages are produced through the action of complex microbial communities. Synthetic biology approaches offer the ability to genetically engineer these communities to improve the properties of these fermented foods. Soy sauce is a fermented condiment with a vast global market. Engineering members of the microbial communities responsible for soy sauce fermentation may therefore lead to the development of improved products. One important property is the colour of soy sauce, with recent evidence pointing to a consumer preference for more lightly-coloured soy sauce products for particular dishes.ResultsHere we show that a bacterial member of the natural soy sauce fermentation microbial community, Bacillus, can be engineered to reduce the ‘browning’ reaction during soy sauce production. We show that two approaches result in ‘de-browning’: engineered consumption of xylose, an important precursor in the browning reaction, and engineered degradation of melanoidins, the major brown pigments in soy sauce. Lastly, we show that these two strategies work synergistically using co-cultures to result in enhanced de-browning.ConclusionsOur results demonstrate the potential of using synthetic biology and metabolic engineering methods for fine-tuning the process of soy sauce fermentation and indeed for many other natural food and beverage fermentations for improved products.

  • Journal article
    Ellis T, 2019,

    What is synthetic genomics anyway?

    , The Biochemist, Vol: 41, Pages: 6-9, ISSN: 0954-982X

    You may have heard of synthetic genomics. This headline-grabbing, high-profile, big science topic is starting to emerge catalysed by the pioneering work of famous names in synthetic biology and biotechnology like George Church and Craig Venter. But what is synthetic genomics and what is it being used for? As a prominent researcher at a recent UK meeting said: “Is it just synthetic biology with bigger bits of DNA?” Well no, not quite…

  • Journal article
    Rajakumar PD, Gower G, Suckling L, Kitney R, McClymont D, Freemont Pet al., 2019,

    Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development

    , Slas Technology, Vol: 24, Pages: 291-297, ISSN: 2472-6303

    Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

  • Journal article
    Witmer K, Fraschka SAK, Vlachou D, Bártfai R, Christophides Get al., 2019,

    Epigenetic regulation underlying Plasmodium berghei gene expression during its developmental transition from host to vector

    , bioRxiv, ISSN: 2045-2322

    ABSTRACT Epigenetic regulation of gene expression is an important attribute in the survival and adaptation of the malaria parasite Plasmodium in its human host. Our understanding of epigenetic regulation of gene expression in Plasmodium developmental stages beyond asexual replication in the mammalian host is sparse. We used chromatin immune-precipitation (ChIP) and RNA sequencing to create an epigenetic and transcriptomic map of the murine parasite Plasmodium berghei development from asexual blood stages to male and female gametocytes, and finally, to ookinetes. We show that heterochromatin 1 (HP1) almost exclusively associates with variantly expressed gene families at subtelomeric regions and remains stable across stages and various parasite lines. Variant expression based on heterochromatic silencing is observed only in very few genes. In contrast, the active histone mark histone 3 Lysine 9 acetylation (H3K9ac) is found between heterochromatin boundaries and occurs as a sharp peak around the start codon for ribosomal protein genes. H3K9ac occupancy positively correlates with gene transcripts in asexual blood stages, male gametocytes and ookinetes. Interestingly, H3K9ac occupancy does not correlate with transcript abundance in female gametocytes. Finally, we identify novel DNA motifs upstream of ookinete-specific genes thought to be involved in transcriptional activation upon fertilization.

  • Journal article
    Perin G, Jones PR, 2019,

    Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels.

    , Current Opinion in Biotechnology, Vol: 57, Pages: 175-182, ISSN: 0958-1669

    Currently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality.

  • Journal article
    Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgard MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, Freemont PSet al., 2019,

    Building a global alliance of biofoundries

    , NATURE COMMUNICATIONS, Vol: 10, Pages: 1-4, ISSN: 2041-1723
  • Journal article
    Gang S, Sharma S, Saraf M, Buck M, Schumacher Jet al., 2019,

    Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method

    , BIO-PROTOCOL, Vol: 9
  • Conference paper
    Kelwick R, Webb AJ, Wang Y, Allan F, Freemont Pet al., 2019,

    ISEV2019 Abstract Book. PT09.10: Protease biomarker detection using functionalised bioplastic-based biosensors

    , ISEV 2019, Publisher: Co-Action Publishing, ISSN: 2001-3078
  • Journal article
    Shaw W, Yamauchi H, Mead J, Gowers G, Bell D, Oling D, Larsson N, Wigglesworth M, Ladds G, Ellis Tet al., 2019,

    Engineering a model cell for rational tuning of GPCR signaling

    , Cell, Vol: 177, Pages: 782-796.e27, ISSN: 0092-8674

    G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond tospecific cues in their environment. However, the relationship between stimulus and response for eachGPCR is difficult to predict due to diversity in natural signal transduction architecture and expression.Using genome engineering in yeast, we here constructed an insulated, modular GPCR signaltransduction system to study how the response to stimuli can be predictably tuned using synthetictools. We delineated the contributions of a minimal set of key components via computational andexperimental refactoring, identifying simple design principles for rationally tuning the dose-response.Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineeredto respond to desired concentrations of peptides, metabolites, and hormones relevant to humanhealth. This work enables rational tuning of cell sensing, while providing a framework to guidereprogramming of GPCR-based signaling in other systems.

  • Journal article
    Debalke S, Habtewold T, Duchateau L, Christophides Get 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: 1756-3305

    BackgroundVector 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.

  • Journal article
    Goey 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: 1369-703X

    Several 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.

  • Journal article
    Stopnitzky E, Still S, Ouldridge TE, Altenberg Let al., 2019,

    Physical limitations of work extraction from temporal correlations.

    , Physical Review E, Vol: 99, ISSN: 1539-3755

    Recently 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.

  • Journal article
    Ruiz VMR, Sousa GL, Sneed SD, Farrant KV, Christophides GK, Povelones Met 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, ISSN: 1932-6203
  • Journal article
    McCarty NS, Shaw WM, Ellis T, Ledesma-Amaro Ret al., 2019,

    Rapid assembly of gRNA arrays via modular cloning in yeast

    , ACS Synthetic Biology, Vol: 8, Pages: 906-910, ISSN: 2161-5063

    CRISPR 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.

  • Journal article
    Boo A, Ellis T, Stan G, 2019,

    Host-aware synthetic biology

    , Current Opinion in Systems Biology, Vol: 14, Pages: 66-72, ISSN: 2452-3100

    Unnatural 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.

  • Journal article
    Kotidis P, Kontoravdi C, 2019,

    Strategic framework for parameterization of cell culture models

    , Processes, Vol: 7, ISSN: 2227-9717

    Global 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.

  • Journal article
    Wen Z, Ledesma-Amaro R, Lin J, Jiang Y, Yang Set al., 2019,

    Improved n-butanol production from Clostridium cellulovorans by integrated metabolic and evolutionary engineering

    , Applied and Environmental Microbiology, Vol: 85, ISSN: 0099-2240

    Clostridium 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.

  • Journal article
    Kuntz J, Thomas P, Stan G-B, Barahona Met 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: 1064-8275

    We 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.

  • Journal article
    Friddin MS, Elani Y, Trantidou T, Ces Oet al., 2019,

    New directions for artificial cells using rapid prototyped biosystems

    , Analytical Chemistry, Vol: 91, Pages: 4921-4928, ISSN: 0003-2700

    Microfluidics 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.

  • Journal article
    Stan G, Ellis T, Boo A, 2019,

    Host-Aware Synthetic Biology

    , Current Opinion in Systems Biology, ISSN: 2452-3100

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