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.
Publications
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Journal articleFreemont PS, Moore S, MacDonald J, et al., 2018,
Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from non-model bacteria
, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E4340-E4349, ISSN: 0027-8424Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
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Journal articleKogenaru M, Isalan M, 2018,
Drug-inducible control of lethality genes: a low background destabilizing domain architecture applied to the Gal4-UAS system in Drosophila
, ACS Synthetic Biology, Vol: 7, Pages: 1496-1506, ISSN: 2161-5063Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteins-of-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. The amount of the DD-tagged fusion protein directly impacts its molecular function. Hence, it is important that the background levels be tightly regulated in the absence of any drug. This is especially true for classes of proteins that function at extremely low levels, such as lethality genes involved in tissue development and certain transcriptional activator proteins. Here, we establish the uninduced background and induction levels for two widely used DDs (FKBP and DHFR) by developing an accurate quantification method. We show that both DDs exhibit functional background levels in the absence of a drug, but each to a different degree. To overcome this limitation, we systematically test a double architecture for these DDs (DD-POI-DD) that completely suppresses the protein’s function in an uninduced state, while allowing tunable functional levels upon adding a drug. As an example, we generate a drug-stabilizable Gal4 transcriptional activator with extremely low background levels. We show that this functions in vivo in the widely used Gal4-UAS bipartite expression system in Drosophila melanogaster. By regulating a cell death gene, we demonstrate that only the low background double architecture enables tight regulation of the lethal phenotype in vivo. These improved tools will enable applications requiring exceptionally tight control of protein function in living cells and organisms.
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Journal articleCeroni F, Boo A, Furini S, et al., 2018,
Burden-driven feedback control of gene expression
, Nature Methods, Vol: 15, Pages: 387-393, ISSN: 1548-7091Cells 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.
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Journal articleDe Porcellinis AJ, Norgaard H, Brey LMF, et al., 2018,
Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp PCC 7002
, Metabolic Engineering, Vol: 47, Pages: 170-183, ISSN: 1096-7176Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic factories for sustainable productions of fuels and chemicals. The Calvin Benson cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants. Previous studies have overexpressed the Calvin Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), in both plants and algae, although their impacts on cyanobacteria have not yet been rigorously studied. Here, we show that overexpression of BiBPase and RuBisCO have distinct impacts on carbon metabolism in the cyanobacterium Synechococcus sp. PCC 7002 through physiological, biochemical, and proteomic analyses. The former enhanced growth, cell size, and photosynthetic O2 evolution, and coordinately upregulated enzymes in the Calvin Benson cycle including RuBisCO and fructose-1,6-bisphosphate aldolase. At the same time it downregulated enzymes in respiratory carbon metabolism (glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase (G6PDH). The content of glycogen was also significantly reduced while the soluble carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and carbon partitioning towards non-storage carbohydrates. In contrast, whilst overexpression of RuBisCO had no measurable impact on growth and photosynthetic O2 evolution, it led to coordinated increase in the abundance of proteins involved in pyruvate metabolism and fatty acid biosynthesis. Our results underpin that singular genetic modifications in the Calvin Benson cycle can have far broader cellular impact than previously expected. These features could be exploited to more efficiently direct carbons towards desired
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Journal articlePerez-Carrasco R, Barnes CP, Schaerli Y, et al., 2018,
Combining a toggle switch and a repressilator within the AC-DC circuit generates distinct dynamical behaviors
, Cell Systems, Vol: 6, Pages: 521-530.e3, ISSN: 2405-4712Although the structure of a genetically encoded regulatory circuit is an important determinant of its function, the relationship between circuit topology and the dynamical behaviors it can exhibit is not well understood. Here, we explore the range of behaviors available to the AC-DC circuit. This circuit consists of three genes connected as a combination of a toggle switch and a repressilator. Using dynamical systems theory, we show that the AC-DC circuit exhibits both oscillations and bistability within the same region of parameter space; this generates emergent behaviors not available to either the toggle switch or the repressilator alone. The AC-DC circuit can switch on oscillations via two distinct mechanisms, one of which induces coherence into ensembles of oscillators. In addition, we show that in the presence of noise, the AC-DC circuit can behave as an excitable system capable of spatial signal propagation or coherence resonance. Together, these results demonstrate how combinations of simple motifs can exhibit multiple complex behaviors.
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Journal articleAvalos JL, Toettcher JE, Lalanne J-B, et al., 2018,
Principles of Systems Biology, No. 28
, CELL SYSTEMS, Vol: 6, Pages: 397-399, ISSN: 2405-4712 -
Journal articleTomazou M, Barahona M, Polizzi K, et al., 2018,
Computational re-design of synthetic genetic oscillators for independent amplitude and frequency modulation
, Cell Systems, Vol: 6, Pages: 508-520.e5, ISSN: 2405-4712To perform well in biotechnology applications, synthetic genetic oscillators must be engineered to allow independent modulation of amplitude and period. This need is currently unmet. Here, we demonstrate computationally how two classic genetic oscillators, the dual-feedback oscillator and the repressilator, can be re-designed to provide independent control of amplitude and period and improve tunability—that is, a broad dynamic range of periods and amplitudes accessible through the input “dials.” Our approach decouples frequency and amplitude modulation by incorporating an orthogonal “sink module” where the key molecular species are channeled for enzymatic degradation. This sink module maintains fast oscillation cycles while alleviating the translational coupling between the oscillator's transcription factors and output. We characterize the behavior of our re-designed oscillators over a broad range of physiologically reasonable parameters, explain why this facilitates broader function and control, and provide general design principles for building synthetic genetic oscillators that are more precisely controllable.
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Journal articleKelly CL, Taylor GM, Hitchcock A, et al., 2018,
A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria.
, ACS Synth Biol, Vol: 7, Pages: 1056-1066Cyanobacteria 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.
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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 articleTomazou M, Stan G-B, 2018,
Portable gene expression guaranteed
, NATURE BIOTECHNOLOGY, Vol: 36, Pages: 313-314, ISSN: 1087-0156 -
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 articlede Lorenzo V, Prather KL, Chen G-Q, et al., 2018,
The power of synthetic biology for bioproduction, remediation and pollution control
, EMBO Reports, Vol: 19, ISSN: 1469-221X -
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 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 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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