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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.

Publications

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  • Journal article
    Weenink T, van der Hilst J, McKiernan R, Ellis Tet al., 2019,

    Design of RNA hairpin modules that predictably tune translation in yeast

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    Modular parts for tuning translation are prevalent in prokaryotic synthetic biology but lacking for eukaryotic synthetic biology. Working in Saccharomyces cerevisiae yeast, we here describe how hairpin RNA structures inserted into the 5′ untranslated region (5′UTR) of mRNAs can be used to tune expression levels by 100-fold by inhibiting translation. We determine the relationship between the calculated free energy of folding in the 5′UTR and in vivo protein abundance, and show that this enables rational design of hairpin libraries that give predicted expression outputs. Our approach is modular, working with different promoters and protein coding sequences, and outperforms promoter mutation as a way to predictably generate a library where a protein is induced to express at a range of different levels. With this new tool, computational RNA sequence design can be used to predictably fine-tune protein production for genes expressed in yeast.

  • Book chapter
    Ouldridge TE, Brittain R, ten Wolde PR, 2018,

    The power of being explicit: demystifying work, heat, and free energy in the physics of computation

    , The Interplay of Thermodynamics and Computation in Both Natural and Artificial Systems
  • Conference paper
    Toczek M, Zielonka D, Zukowska P, Marcinkowski JT, Kutryb-Zajac B, Slominska E, Isalan M, Mielcarek M, Smolenski RTet al., 2018,

    An altered nucleotide metabolism as a novel mechanism leading to Huntington disease related cardiomyopathy

    , Purines 2018 Basic and Translational Science on Purinergic Signaling and its Components for a Healthy and Better World, Publisher: Springer Verlag, Pages: S53-S53, ISSN: 1573-9538
  • Journal article
    Kyrou K, Hammond AM, Galizi R, Kranjc N, Burt A, Beaghton AK, Nolan T, Crisanti Aet al., 2018,

    A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

    , Nature Biotechnology, Vol: 36, Pages: 1062-1066, ISSN: 1087-0156

    In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.

  • Journal article
    Kelly CL, Harris AWK, Steel H, Hancock EJ, Heap JT, Papachristodoulou Aet al., 2018,

    Synthetic negative feedback circuits using engineered small RNAs

    , Nucleic Acids Research, Vol: 46, Pages: 9875-9889, ISSN: 0305-1048

    Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

  • Journal article
    Gorochowski TE, Ellis T, 2018,

    Designing efficient translation

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 934-935, ISSN: 1087-0156
  • Journal article
    Trantidou T, Dekker L, Polizzi K, Ces O, Elani Yet al., 2018,

    Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

    , Interface Focus, Vol: 8, ISSN: 2042-8901

    The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

  • Journal article
    Waters AJ, Capriotti P, Gaboriau DCA, Papathanos PA, Windbichler Net al., 2018,

    Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

    , Scientific Reports, Vol: 8, ISSN: 2045-2322

    The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

  • Journal article
    Yunus IS, Wichmann J, Wördenweber R, Lauersen KJ, Kruse O, Jones PRet al., 2018,

    Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel

    , Metabolic Engineering, Vol: 49, Pages: 201-211, ISSN: 1096-7176

    Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synt

  • Conference paper
    Toczek M, Zielonka D, Marcinkowski J, Isalan M, Smolenski R, Mielcarek Met al.,

    An altered metabolism of nucleotides leads to huntington’s disease related cardiomyopathy

    , EHDN Plenary Meeting, Publisher: BMJ Publishing Group, Pages: A13-A13, ISSN: 1468-330X
  • Conference paper
    Puch A, Witkowski G, Isalan M, Mielcarek M, Zielonka Det al., 2018,

    A frequency of concomitant disorders in presymptomatic huntington’s disease patients

    , EHDN Plenary Meeting 2018, Publisher: BMJ Publishing Group, Pages: A42-A43, ISSN: 1468-330X
  • Journal article
    Schaerli Y, Jiménez A, Duarte JM, Mihajlovic L, Renggli J, Isalan M, Sharpe J, Wagner Aet al., 2018,

    Synthetic circuits reveal how mechanisms of gene regulatory networks constrain evolution

    , Molecular Systems Biology, Vol: 14, ISSN: 1744-4292

    Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe—a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

  • Journal article
    Aw R, McKay P, Shattock R, Polizzi Ket al., 2018,

    A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization

    , Protein Expression and Purification, Vol: 149, Pages: 43-50, ISSN: 1046-5928

    Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

  • Journal article
    Yunus IS, Jones PR, 2018,

    Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols

    , Metabolic Engineering, Vol: 49, Pages: 59-68, ISSN: 1096-7176

    Cyanobacteria can directly channel atmospheric CO2 into a wide range of versatile carbon products such as fatty acids and fatty alcohols with applications including fuel, cosmetics, and health products. Works on alcohol production in cyanobacteria have so far focused on either long (C12-C18) or short (C2-C4) chain-length products. In the present work, we report the first synthetic pathway for 1-octanol (C8) biosynthesis in Synechocystis sp. PCC 6803, employing a carboxylic acid reductase and C8-preferring fatty acyl-ACP thioesterase. The first engineered strain produced 1-octanol but exhibited poor productivity and cellular health issues. We therefore proceeded to systematically optimize the strain and cultivation conditions in order to understand what the limiting factors were. The identification of optimal promoters and ribosomal binding sites, in combination with isopropyl myristate solvent overlay, resulted in a combined (C8-OH and C10-OH) titer of more than 100 mg/L (a 25-fold improvement relative to the first engineered strain) and a restoration of cellular health. Additionally, more than 905 mg/L 1-octanol was produced when the strain expressing sfp (phosphopantetheinyl transferase) and car (carboxylic acid reductase) was fed with octanoic acid. A combination of feeding experiments and protein quantification indicated that the supply of octanoic acid from the introduced thioesterase, and possibly also native fatty acid synthesis pathway, were the main bottlenecks of the pathway.

  • Journal article
    Oling D, Lawenius L, Shaw W, Clark S, Kettleborough R, Ellis T, Larsson N, Wigglesworth Met al., 2018,

    Large Scale Synthetic Site Saturation GPCR Libraries Reveal Novel Mutations That Alter Glucose Signaling

    , ACS SYNTHETIC BIOLOGY, Vol: 7, Pages: 2317-2321, ISSN: 2161-5063
  • Journal article
    Ceroni F, Ellis T, 2018,

    The challenges facing synthetic biology in eukaryotes

    , Nature Reviews Molecular Cell Biology, Vol: 19, Pages: 481-482, ISSN: 1471-0072

    Synthetic biology is maturing into a true engineering discipline for model microorganisms, but remains far from straightforward for most eukaryotes. Here, we outline the key challenges facing those trying to engineer biology across eukaryota and suggest areas of focus that will aid future progress.

  • Journal article
    Kylilis N, Tuza ZA, Stan G, Polizzi KMet al., 2018,

    Tools for engineering coordinated system behaviour in synthetic microbial consortia

    , Nature Communications, Vol: 9, ISSN: 2041-1723

    Advancing synthetic biology to the multicellular level requires the development of multiple cell-to-cell communication channels that propagate information with minimal signal interference. The development of quorum-sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on cognate acyl-homoserine lactone (AHL)/transcription factor pairs, while the use of non-cognate pairs as a design feature has received limited attention. Here, we demonstrate a large library of AHL-receiver devices, with all cognate and non-cognate chemical signal interactions quantified, and we develop a software tool that automatically selects orthogonal communication channels. We use this approach to identify up to four orthogonal channels in silico, and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels is an enabling step that facilitates the design of synthetic consortia for applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation and spatial organisation.

  • Journal article
    Liu D, Mannan AA, Han Y, Oyarzun DA, Zhang Fet al., 2018,

    Dynamic metabolic control: towards precision engineering of metabolism

    , Journal of Industrial Microbiology and Biotechnology, Vol: 45, Pages: 535-543, ISSN: 1367-5435

    Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the “push–pull-block” strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.

  • Journal article
    Beal J, Haddock-Angelli T, Baldwin G, Gershater M, Dwijayanti A, Storch M, de Mora K, Lizarazo M, Rettberg Ret al., 2018,

    Quantification of bacterial fluorescence using independent calibrants

    , PLoS ONE, Vol: 13, ISSN: 1932-6203

    Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.

  • Journal article
    Enrico Bena C, Grob A, Isalan M, Bosia C, Ceroni Fet al., 2018,

    Commentary: Synthetic Addiction Extends the Productive Life Time of Engineered Escherichia coli Populations

    , Frontiers in Bioengineering and Biotechnology, Vol: 6, ISSN: 2296-4185

    A commentary on Synthetic addiction extends the productive life time of engineered Escherichia coli populations by Rugbjerg, P., Sarup-Lytzen, K., Nagy, M., and Sommer, M. O. A. (2018). Proc. Natl. Acad. Sci. U.S.A. 115, 2347–2352. doi: 10.1073/pnas.1718622115Bioproduction is the process of producing added-value chemicals on large-scale using cells as biological factories. Cellular burden represents a significant problem in the scaling of fermentation processes from proof-of-concept to long-term cultures, as the load of heterologous gene expression and depletion of the cell intracellular resources cause unpredictable cellular physiological changes that can lead to decreased growth and lower production yields (Borkowski et al., 2016; Liu et al., 2018). One possible cause of the observed decreased bioproduct recovery in many bioprocessing applications is the accumulation of mutations in the employed genetic program. These mutations often lead to loss of production and rise of non-producing populations that grow better and easily overtake the growth of producing cells (Rugbjerg et al., 2018b).In a recent paper in PNAS, Rugbjerg et al. (2018b) developed a strategy to limit the enrichment of non-producing cell populations in bioproduction-employed cell cultures by placing the genes for key growth intermediates under the control of a promoter responsive to the bioproduct being made. This strategy known as product addiction was tested in E. coli engineered to produce mevalonic acid in long-term cultivations (Figure 1).

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