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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
    Avalos JL, Toettcher JE, Lalanne J-B, Li G-W, Gomes ALC, Johns NI, Wang HH, Ellis T, Stan G-B, Mure LS, Panda S, Cooper HM, Fernandez-Martinez J, Rout MP, Akey CW, Kim SJ, Sali A, Bastarache L, Denny JCet al., 2018,

    Principles of Systems Biology, No. 28

    , CELL SYSTEMS, Vol: 6, Pages: 397-399, ISSN: 2405-4712
  • Journal article
    Kelly CL, Taylor GM, Hitchcock A, Torres-Méndez A, Heap JTet al., 2018,

    A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria.

    , ACS Synth Biol, Vol: 7, Pages: 1056-1066

    Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

  • Journal article
    Borkowski O, Bricio C, Murgiano M, Rothschild-Mancinelli B, Stan G, Ellis Tet al., 2018,

    Cell-free prediction of protein expression costs for growing cells

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

    Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any given gene is a major challenge, as multiple processes and factors combine to determine translation efficiency. To enable prediction of the cost of gene expression in bacteria, we describe here a standard cell-free lysate assay that provides a relative measure of resource consumption when a protein coding sequence is expressed. These lysate measurements can then be used with a computational model of translation to predict the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.

  • Journal article
    Fonseca P, Romano F, Schreck JS, Ouldridge TE, Doye JPK, Louis AAet al., 2018,

    Multi-scale coarse-graining for the study of assembly pathways in DNA-brick self assembly

    , Journal of Chemical Physics, Vol: 148, ISSN: 0021-9606

    Inspired by recent successes using single-stranded DNA tiles to producecomplex structures, we develop a two-step coarse-graining approach that usesdetailed thermodynamic calculations with oxDNA, a nucleotide-based model ofDNA, to parametrize a coarser kinetic model that can reach the time and lengthscales needed to study the assembly mechanisms of these structures. We test themodel by performing a detailed study of the assembly pathways for atwo-dimensional target structure made up of 334 unique strands each of whichare 42 nucleotides long. Without adjustable parameters, the model reproduces acritical temperature for the formation of the assembly that is close to thetemperature at which assembly first occurs in experiments. Furthermore, themodel allows us to investigate in detail the nucleation barriers and thedistribution of critical nucleus shapes for the assembly of a single targetstructure. The assembly intermediates are compact and highly connected(although not maximally so) and classical nucleation theory provides a good fitto the height and shape of the nucleation barrier at temperatures close towhere assembly first occurs.

  • Journal article
    Tomazou M, Stan G-B, 2018,

    Portable gene expression guaranteed

    , NATURE BIOTECHNOLOGY, Vol: 36, Pages: 313-314, ISSN: 1087-0156
  • Journal article
    Grob A, Marbiah MM, Isalan M, 2018,

    Functional insulator scanning of CpG islands to identify regulatory regions of promoters using CRISPR

    , Methods in Molecular Biology, Vol: 1766, Pages: 285-301, ISSN: 1940-6029

    The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (β-globin) insulator, 5′HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via “insulator scanning.” Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.

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

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

    , Protein Expression and Purification, ISSN: 1046-5928
  • Journal article
    Ceroni F, Boo A, Furini S, Gorochowski T, Ladak Y, Awan A, Gilbert C, Stan G, Ellis Tet al., 2018,

    Burden-driven feedback control of gene expression

    , Nature Methods, Vol: 15, Pages: 387-393, ISSN: 1548-7091

    Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

  • Journal article
    de Lorenzo V, Prather KL, Chen G-Q, O'Day E, von Kameke C, Oyarzún DA, Hosta-Rigau L, Alsafar H, Cao C, Ji W, Okano H, Roberts RJ, Ronaghi M, Yeung K, Zhang F, Lee SYet al., 2018,

    The power of synthetic biology for bioproduction, remediation and pollution control

    , EMBO Reports, Vol: 19, ISSN: 1469-221X
  • Journal article
    Pothoulakis G, Ellis T, 2018,

    Construction of hybrid regulated mother-specific yeast promoters for inducible differential gene expression

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

    Engineered promoters with predefined regulation are a key tool for synthetic biology that enable expression on demand and provide the logic for genetic circuits. To expand the availability of synthetic biology tools for S. cerevisiae yeast, we here used hybrid promoter engineering to construct tightly-controlled, externally-inducible promoters that only express in haploid mother cells that have contributed a daughter cell to the population. This is achieved by combining elements from the native HO promoter and from a TetR-repressible synthetic promoter, with the performance of these promoters characterized by both flow cytometry and microfluidics-based fluorescence microscopy. These new engineered promoters are provided as an enabling tool for future synthetic biology applications that seek to exploit differentiation within a yeast population.

  • Journal article
    Cox R, Madsen C, McLaughlin J, Nguyen T, Roehner N, Bartley B, Bhatia S, Bissell M, Clancy K, Gorochowski T, Grunberg R, Luna A, Le Novere N, Pocock M, Sauro H, Sexton J, Stan G, Tabor J, Voigt C, Zundel Z, Myers C, Beal J, Wipat Aet al., 2018,

    Synthetic Biology Open Language Visual (SBOL Visual) Version 2.0

    , Journal of Integrative Bioinformatics, Vol: 15, ISSN: 1613-4516

    People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.0 of SBOL Visual, which builds on the prior SBOL Visual 1.0 standard by expanding diagram syntax to include functional interactions and molecular species, making the relationship between diagrams and the SBOL data model explicit, supporting families of symbol variants, clarifying a number of requirements and best practices, and significantly expanding the collection of diagram glyphs.

  • Journal article
    Elani Y, Trantidou T, Wylie D, Dekker L, Polizzi K, Law R, Ces Oet al., 2018,

    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

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

    There is increasing interest in constructing artificial cells by functionalisinglipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limitedin comparison to their biologicalcounterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger syntheticcell assembly. Using microfluidic technologies to construct such hybrid systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, whilethe cellacts as a bioreactor module that processes encapsulated feedstock which is further processedby a synthetic enzymatic cascadeco-encapsulated in the vesicle.

  • Journal article
    Jonas FRH, Royle KE, Aw R, Stan G, Polizzi KMet al., 2018,

    Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling

    , Synthetic and Systems Biotechnology, Vol: 3, Pages: 64-75, ISSN: 2405-805X

    Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays. In this study, we have used a combination of fluorescent reporters, flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis. Under low ER stress, asymmetric division leaves daughter cells with an ER deficiency, necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth. Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells. These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.

  • Journal article
    Broedel AK, Isalan M, 2018,

    Trp-ing upon new repressors

    , Nature Chemical Biology, Vol: 14, Pages: 328-329, ISSN: 1552-4450

    Bioengineers have used directed evolution to generate a new family of synthetic transcription factors based on the tryptophan repressor. The evolved repressor family enables researchers to build new gene circuits for biomedical applications.

  • Journal article
    Heide C, Ces O, Polizzi K, Kontoravdi Cet al.,

    Creating cell-free protein synthesis factories

    , Pharmaceutical Bioprocessing, ISSN: 2048-9145
  • Journal article
    Heide C, Ces O, Polizzi K, Kontoravdi Cet al.,

    Creating cell-free protein synthesis factories

    , Pharmaceutical Bioprocessing, ISSN: 2048-9145
  • Journal article
    Niehus X, Crutz-LeCoq A-M, Sandoval G, Nicaud J-M, Ledesma Amaro Ret al., 2018,

    Engineering Yarrowia lipolytica to enhance lipid production from lignocellulosic materials

    , Biotechnology for Biofuels, Vol: 11, ISSN: 1754-6834

    Background: Yarrowia lipolytica is a common biotechnological chassis for the production of lipids, which are the pre‑ferred feedstock for the production of fuels and chemicals. To reduce the cost of microbial lipid production, inexpen‑sive carbon sources must be used, such as lignocellulosic hydrolysates. Unfortunately, lignocellulosic materials oftencontain toxic compounds and a large amount of xylose, which cannot be used by Y. lipolytica.Results: In this work, we engineered this yeast to efciently use xylose as a carbon source for the productionof lipids by overexpressing native genes. We further increased the lipid content by overexpressing heterologousgenes to facilitate the conversion of xylose-derived metabolites into lipid precursors. Finally, we showed that theseengineered strains were able to grow and produce lipids in a very high yield (lipid content = 67%, titer = 16.5 g/L,yield = 3.44 g/g sugars, productivity 1.85 g/L/h) on a xylose-rich agave bagasse hydrolysate in spite of toxiccompounds.Conclusions: This work demonstrates the potential of metabolic engineering to reduce the costs of lipid productionfrom inexpensive substrates as source of fuels and chemicals.

  • Journal article
    Pothoulakis G, Ellis T, 2018,

    Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype

    , Communications Biology, Vol: 1, ISSN: 2399-3642

    Pseudohyphal growth is a multicellular phenotype naturally performed by wild budding yeast cells in response to stress. Unicellular yeast cells undergo gross changes in their gene regulation and elongate to form branched filament structures consisting of connected cells. Here, we construct synthetic gene regulation systems to enable external induction of pseudohyphal growth in Saccharomyces cerevisiae. By controlling the expression of the natural PHD1 and FLO8 genes we are able to trigger pseudohyphal growth in both diploid and haploid yeast, even in different types of rich media. Using this system, we also investigate how members of the BUD gene family control filamentation in haploid cells. Finally, we employ a synthetic genetic timer network to control pseudohyphal growth and further explore the reversibility of differentiation. Our work demonstrates that synthetic regulation can exert control over a complex multigene phenotype and offers opportunities for rationally modifying the resulting multicellular structure.

  • Journal article
    Mordaka PM, Heap JT, 2018,

    Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates

    , ACS Synthetic Biology, Vol: 7, Pages: 672-681, ISSN: 2161-5063

    Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus −35 and −10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthl was 79% or 58%, instead of 25% as before. Synthetic promoters from these “stringent” libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected “stringency” of promoter sequences in Clostridium, not reported previously.

  • Book chapter
    Lai H-E, Moore S, Polizzi K, Freemont Pet al., 2018,

    EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

    , Pages: 429-444

    Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

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