Research
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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 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 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 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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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 articleHazel P, Kroll SHB, Bondke A, et al., 2018,
Corrigendum: Inhibitor selectivity for cyclin-dependent kinase 7: a structural, thermodynamic, and modelling study
, ChemMedChem, Vol: 13, Pages: 207-207, ISSN: 1860-7187 -
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: 672-681, ISSN: 2161-5063Collections 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 articleFreemont PS, Salih O, He S, et al., 2018,
Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa
, Structure, Vol: 26, Pages: 329-336.e3, ISSN: 0969-2126Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
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Book chapterLai H-E, Moore S, Polizzi K, et al., 2018,
EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.
, Pages: 429-444Development 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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Journal articleKhara DC, Schreck JS, Tomov TE, et al., 2017,
DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size
, Nucleic Acids Research, Vol: 46, Pages: 1553-1561, ISSN: 0305-1048We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions.
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Journal articleClaesen S, Stone A, van Rossum M, et al., 2017,
Comprehensive web-based broker for bio-technology design and manufacturing
, Engineering Biology, Vol: 1, Pages: 100-102, ISSN: 2398-6182Synthetic biology, particularly in relation to characterisation experiments relating to the description of bio-parts frequently involves the use of a wide range of equipment, including, for example, plate reader's, flow cytometers, and mass spectrometers. This equipment is often from multiple manufacturers. The study describes broker technology that has been developed which has the ability to connect multiple types of equipment into a common information environment; the connectivity from the databases and equipment is achieved using Visbion's ‘cube’ technology that involves military specification encryption for data security. The broker technology uses a new, developing standard, Digital Imaging and Communication in Medicine (DICOM)-SB, that is based on the highly successful international standard for biomedicine, DICOM. The broker uses a version of the DICOM data model that has been specifically designed for synthetic biology and, in particular, characterisation data.
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Journal articleDavidchack RL, Ouldridge TE, Tretyakov MV, 2017,
Geometric integrator for Langevin systems with quaternion-based rotational degrees of freedom and hydrodynamic interactions
, Journal of Chemical Physics, Vol: 147, ISSN: 0021-9606We introduce new Langevin-type equations describing the rotational andtranslational motion of rigid bodies interacting through conservative andnon-conservative forces, and hydrodynamic coupling. In the absence ofnon-conservative forces the Langevin-type equations sample from the canonicalensemble. The rotational degrees of freedom are described using quaternions,the lengths of which are exactly preserved by the stochastic dynamics. For theproposed Langevin-type equations, we construct a weak 2nd order geometricintegrator which preserves the main geometric features of the continuousdynamics. A number of numerical experiments are presented to illustrate boththe new Langevin model and the numerical method for it.
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Journal articleBroedel AK, Isalan M, Jaramillo A, 2017,
Engineering of biomolecules by bacteriophage directed evolution
, Current Opinion in Biotechnology, Vol: 51, Pages: 32-38, ISSN: 0958-1669Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein–protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes.
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Journal articleHancock E, Ang J, Papachristodoulou A, et al., 2017,
The interplay between feedback and buffering in homeostasis
, Cell Systems, Vol: 5, Pages: 498-508.e23, ISSN: 2405-4712Feedback and buffering---the use of reservoirs of molecules to maintain molecular concentrations---are the primary mechanisms for robust regulation in biochemical processes. The universal principles behind their combined effect, however, have not been studied before. Here, we determine the fundamental forms of cooperation and tradeoff between buffering and feedback. We find that negative feedback regulates slow-changing components of time-varying signals, while buffering regulates fast-changing components, consistent with observations of both ATP and pH regulation. We further find that buffering stabilizes feedback and improves its effectiveness, but also introduces molecular noise. In addition, we show that rapid-acting buffering imparts negative derivative feedback, while slow-acting buffering can result in feedforward filtering of specific signals; both are control strategies widely used in technology. Finally, we discover an empirical cross-species relationship between feedback in glycolysis and ATP buffering that is consistent with our findings.
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Journal articleOuldridge TE, 2017,
The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamics
, Natural Computing, Vol: 17, Pages: 3-29, ISSN: 1567-7818Improved understanding of molecular systems has only emphasised thesophistication of networks within the cell. Simultaneously, the advance ofnucleic acid nanotechnology, a platform within which reactions can beexquisitely controlled, has made the development of artificial architecturesand devices possible. Vital to this progress has been a solid foundation in thethermodynamics of molecular systems. In this pedagogical review andperspective, I will discuss how thermodynamics determines both the overallpotential of molecular networks, and the minute details of design. I will thenargue that, in turn, the need to understand molecular systems is helping todrive the development of theories of thermodynamics at the microscopic scale.
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Journal articleRoyle KE, Polizzi KM, 2017,
A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris
, Scientific Reports, Vol: 7, ISSN: 2045-2322Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.
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Journal articleWintle BC, Boehm CR, Rhodes C, et al., 2017,
A transatlantic perspective on 20 emerging issues in biological engineering
, eLife, Vol: 6, ISSN: 2050-084XAdvances in biological engineering are likely to have substantial impacts on global society. To explorethese potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunitiesand risks presented by biological engineering. We first identified 70 potential issues, and then used an iterativeprocess to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to berelatively unknown outside the field of biological engineering. The issues identified may be of interest toresearchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.
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Journal articlePark YK, Nicaud JM, Ledesma Amaro R, 2017,
The engineering potential of Rhodosporidium toruloides as a workhorse for biotechnological applications
, Trends in Biotechnology, Vol: 36, Pages: 304-317, ISSN: 0167-7799Moving our society towards a bioeconomy requires efficient and sustainable microbial production of chemicals and fuels. Rhodotorula (Rhodosporidium) toruloides is a yeast that naturally synthesizes substantial amounts of specialty chemicals and has been recently engineered to (i) enhance its natural production of lipids and carotenoids, and (ii) produce novel industrially relevant compounds. The use of R. toruloides by companies and research groups has exponentially increased in recent years as a result of recent improvements in genetic engineering techniques and the availability of multiomics information on its genome and metabolism. This review focuses on recent engineering approaches in R. toruloides for bioproduction and explores its potential as a biotechnological chassis.
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