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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 articleBorkowski O, Bricio C, Murgiano M, et al., 2018,
Cell-free prediction of protein expression costs for growing cells
, Nature Communications, Vol: 9, Pages: 1-11, ISSN: 2041-1723Translating 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.
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Journal articleFonseca P, Romano F, Schreck JS, et al., 2018,
Multi-scale coarse-graining for the study of assembly pathways in DNA-brick self assembly
, Journal of Chemical Physics, Vol: 148, ISSN: 0021-9606Inspired 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.
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Journal articleGrob 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: 1940-6029The 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.
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Journal articleTomazou M, Stan G-B, 2018,
Portable gene expression guaranteed
, NATURE BIOTECHNOLOGY, Vol: 36, Pages: 313-314, ISSN: 1087-0156 -
Journal articleAw R, McKay P, Shattock R, et 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, ISSN: 1046-5928 -
Journal articlePothoulakis G, Ellis T, 2018,
Construction of hybrid regulated mother-specific yeast promoters for inducible differential gene expression
, PLoS ONE, Vol: 13, ISSN: 1932-6203Engineered 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.
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Journal articleCox R, Madsen C, McLaughlin J, et al., 2018,
Synthetic Biology Open Language Visual (SBOL Visual) Version 2.0
, Journal of Integrative Bioinformatics, Vol: 15, ISSN: 1613-4516People 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.
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Journal articleHindley JW, Elani Y, McGilvery CM, et al., 2018,
Light-triggered enzymatic reactions in nested vesicle reactors
, Nature Communications, Vol: 9, Pages: 1-6, ISSN: 2041-1723Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.
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Journal articleElani Y, Trantidou T, Wylie D, et al., 2018,
Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules
, Scientific Reports, Vol: 8, Pages: 1-8, ISSN: 2045-2322There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. 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 synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic 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, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
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Journal articleJonas FRH, Royle KE, Aw R, et 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: 2405-805XAdaptation 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.
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Journal articleSainz de Murieta I, Bultelle M, Kitney R, 2018,
Data model for biopart datasheets
, Engineering Biology, Vol: 2, Pages: 7-18, ISSN: 2398-6182This study introduces a new data model, based on the DICOM-SB (see glossary of terms for definition of acronyms) standard for synthetic biology, that is capable of describing/incorporating the data, metadata and ancillary information from detailed characterisation experiments - to present DNA components (bioparts) in datasheets. The data model offers a standardised mechanism to associate bioparts with data and information about component performance - in a particular biological context (or a range of contexts, e.g. chassis). The data model includes the raw, experimental data for each characterisation run, and the protocol details needed to reliably reproduce the experiment. In addition, it provides metrics (e.g. relative promoter units, synthesis/growth rates etc.) that constitute the main content of a biopart datasheet. The data model has been developed to directly link to DICOM-SB, but also to be compatible with existing data standards, e.g. SBOL and SBML. It has been implemented within the latest version of the API that enables access to the SynBIS information system. The work should contribute significantly to the current standardisation effort in synthetic biology. The standard data model for datasheets is seen as a necessary step towards effective interoperability between part repositories, and between repositories and BioCAD applications.
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Journal articleGang S, Sarah M, Waite C, et al., 2018,
Mutualism between Klebsiella SGM 81 and Dianthus caryophyllus in modulating root plasticity and rhizospheric bacterial density
, Plant and Soil, Vol: 424, Pages: 273-288, ISSN: 0032-079XAimsDianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.MethodsipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.ResultsInoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high
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Journal articleBroedel AK, Isalan M, 2018,
Trp-ing upon new repressors
, Nature Chemical Biology, Vol: 14, Pages: 328-329, ISSN: 1552-4450Bioengineers 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.
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Journal articleMordaka 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: 672-681Collections 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.
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Journal articleGoey CH, Bell D, Kontoravdi K, 2018,
Mild hypothermic culture conditions impact residual host cell protein composition post-protein a chromatography
, mAbs, Vol: 10, Pages: 476-487, ISSN: 1942-0862Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry experiment with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.
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Journal articleHeide C, Ces O, Polizzi K, et al., 2018,
Creating cell-free protein synthesis factories
, Pharmaceutical Bioprocessing, Vol: 6, Pages: 3-6, ISSN: 2048-9145 -
Journal articlePapathanos PA, Windbichler N, 2018,
Redkmer: An assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases
, The Crispr Journal, Vol: 1, Pages: 88-98, ISSN: 2573-1599CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the charact
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Journal articleSellés Vidal L, Kelly CL, Mordaka PM, et al., 2018,
Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application.
, Biochim Biophys Acta Proteins Proteom, Vol: 1866, Pages: 327-347, ISSN: 1570-9639NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)+. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.
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Journal articleNiehus X, Crutz-LeCoq A-M, Sandoval G, et al., 2018,
Engineering Yarrowia lipolytica to enhance lipid production from lignocellulosic materials
, Biotechnology for Biofuels, Vol: 11, ISSN: 1754-6834Background: 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.
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Journal articlePothoulakis G, Ellis T, 2018,
Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype
, Communications Biology, Vol: 1, ISSN: 2399-3642Pseudohyphal 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.
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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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