Publications
57 results found
Elani Y, Seddon J, 2023, What it means to be alive: a synthetic cell perspective, Interface Focus, Vol: 13, Pages: 1-3, ISSN: 2042-8898
Advances in bottom-up synthetic biology offer the exciting—albeit contentious—prospect of transitioning bio-science researchers from passive observers of life to potential creators of it. Synthetic cells closely emulate the attributes of their biological counterparts. These rationally designed microsystems exhibit emergent properties and life-like functionalities. They can therefore be used as simplified cell models to decipher the rules of life, and as programmable biologically powered micromachines for application in healthcare and biotechnology more broadly. While there is a consensus that current synthetic cells are not yet ‘living’, the question of what defines ‘aliveness’ is gaining increasing relevance. Exploring this concept necessitates a multidisciplinary approach, where scientists from across domains in the physical, life, engineering and social sciences participate in community-level discussions, together with the acceptance of a set of criteria which defines a living system. Achieving a widely accepted definition of ‘living’ represents a possible mission-oriented endpoint to the synthetic cell endeavour, uniting the community towards a common goal. As the field evolves, researchers must address regulatory, ethical, societal and public perception implications, while fostering collaborative efforts to harness the transformative potential of synthetic cells.
Allen ME, Hindley J, O'Toole N, et al., 2023, Biomimetic Behaviours in Hydrogel Artificial Cells through Embedded Organelles, Proceedings of the National Academy of Sciences of USA, Vol: 120, ISSN: 0027-8424
Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom–up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.
Pilkington C, Contini C, Barritt J, et al., 2023, A microfluidic platform for the controlled synthesis of architecturally complex liquid crystalline nanoparticles, Scientific Reports, Vol: 13, Pages: 1-14, ISSN: 2045-2322
Soft-matter nanoparticles are of great interest for their applications in biotechnology, therapeutic delivery, and in vivo imaging. Underpinningthis is their biocompatibility, potential for selective targeting, attractive pharmacokinetic properties, and amenability to downstreamfunctionalisation. Morphological diversity inherent to soft-matter particles can give rise to enhanced functionality. However, this diversityremains untapped in clinical and industrial settings, and only the simplest of particle architectures (spherical lipid vesicles and lipid/polymernanoparticles (LNPs)) have been exploited. To address this, we have designed a scalable microfluidic hydrodynamic focusing (MHF)technology for the controllable, rapid, and continuous production of lyotropic liquid crystalline (LLC) nanoparticles (both cubosomes andhexosomes), colloidal dispersions of higher-order lipid assemblies with intricate internal structures of 3-D and 2-D symmetry. These particleshave been proposed as the next generation of soft-matter nano-carriers, with unique fusogenic and physical properties. Crucially, unlikealternative approaches, our microfluidic method gives control over LLC size, a feature we go on to exploit in a fusogenic study with modelcell membranes, where a dependency on particle diameter is evident. We believe our platform has the potential to serve as a tool for futurestudies involving non-lamellar soft nanoparticles, and anticipate it allowing for the rapid prototyping of LLC particles of diverse functionality,paving the way toward their eventual uptake at an industrial level.
Raguseo F, Huyghebaert A, Li J, et al., 2023, The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes
<jats:p>Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is the expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigated the formation of nucleic-acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of the diseases. We observed significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associated to the formation of multimolecular G-quadruplexes (mG4s), using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions led to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validated the biological relevance of our findings by demonstrating the ability of a G4-selective fluorescent probe to penetrate C9orf72 mutant human motor neurons derived from ALS patients, which revealed clear fluorescent signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation related ALS and FTD.</jats:p>
Gispert Contamina I, Hindley J, Pilkington C, et al., 2022, Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation, Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-10, ISSN: 0027-8424
Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.
Allen ME, Hindley JW, Baxani DK, et al., 2022, Hydrogels as functional components in artificial cell systems, Nature Reviews Chemistry, Vol: 6, Pages: 562-578, ISSN: 2397-3358
Recent years have seen substantial efforts aimed at constructing artificial cells from various molecular components with the aim of mimicking the processes, behaviours and architectures found in biological systems. Artificial cell development ultimately aims to produce model constructs that progress our understanding of biology, as well as forming the basis for functional bio-inspired devices that can be used in fields such as therapeutic delivery, biosensing, cell therapy and bioremediation. Typically, artificial cells rely on a bilayer membrane chassis and have fluid aqueous interiors to mimic biological cells. However, a desire to more accurately replicate the gel-like properties of intracellular and extracellular biological environments has driven increasing efforts to build cell mimics based on hydrogels. This has enabled researchers to exploit some of the unique functional properties of hydrogels that have seen them deployed in fields such as tissue engineering, biomaterials and drug delivery. In this Review, we explore how hydrogels can be leveraged in the context of artificial cell development. We also discuss how hydrogels can potentially be incorporated within the next generation of artificial cells to engineer improved biological mimics and functional microsystems.
Zubaite G, Hindley JW, Ces O, et al., 2022, Dynamic reconfiguration of subcompartment architectures in artificial cells., ACS Nano, Vol: 16, ISSN: 1936-0851
Artificial cells are minimal structures constructed from biomolecular building blocks designed to mimic cellular processes, behaviors, and architectures. One near-ubiquitous feature of cellular life is the spatial organization of internal content. We know from biology that organization of content (including in membrane-bound organelles) is linked to cellular functions and that this feature is dynamic: the presence, location, and degree of compartmentalization changes over time. Vesicle-based artificial cells, however, are not currently able to mimic this fundamental cellular property. Here, we describe an artificial cell design strategy that addresses this technological bottleneck. We create a series of artificial cell architectures which possess multicompartment assemblies localized either on the inner or on the outer surface of the artificial cell membrane. Exploiting liquid-liquid phase separation, we can also engineer spatially segregated regions of condensed subcompartments attached to the cell surface, aligning with coexisting membrane domains. These structures can sense changes in environmental conditions and respond by reversibly transitioning from condensed multicompartment layers on the membrane surface to a dispersed state in the cell lumen, mimicking the dynamic compartmentalization found in biological cells. Likewise, we engineer exosome-like subcompartments that can be released to the environment. We can achieve this by using two types of triggers: chemical (addition of salts) and mechanical (by pulling membrane tethers using optical traps). These approaches allow us to control the compartmentalization state of artificial cells on population and single-cell levels.
Contini C, Hu W, Elani Y, 2022, Manufacturing polymeric porous capsules, Chemical Communications, Vol: 58, Pages: 4409-4419, ISSN: 1359-7345
Polymeric porous capsules represent hugely promising systems that allow a size-selective through-shell material exchange with their surroundings. They have vast potential in applications ranging from drug delivery and chemical microreactors to artificial cell science and synthetic biology. Due to their porous core-shell structure, polymeric porous capsules possess an enhanced permeability that enables the exchange of small molecules while retaining larger compounds and macromolecules. The cross-capsule transfer of material is regulated by their pore size cut-off, which depends on the molecular composition and adopted fabrication method. This review outlines the main strategies adopted for manufacturing polymeric porous capsules to provide some practical guidance for designing polymeric capsules with controlled pore size.
Monck C, Elani Y, Ceroni F, 2022, Cell-free protein synthesis: biomedical applications and future perspectives, Chemical Engineering Research and Design, Vol: 177, Pages: 653-658, ISSN: 0263-8762
The use of cell-free protein synthesis (CFPS) has become increasingly widespread in synthetic biology over recent years, providing an effective platform for the study and engineering of cellular processes. The versatility and portability of CFPS systems have also boosted their potential for usage outside of the laboratory in a wide number of applications, from construct prototyping to bioproduction. CFPS is particularly well suited to biomedical applications, such as the production of clinical molecules and vaccines. It can also be integrated with additional technologies such as microfluidics and liposomal encapsulation to provide a new route for on-demand therapeutic expression. In this review we outline the key features of CFPS that make it a powerful platform for biomedical applications. We also discuss existing limitations with respect to the use of CFPS in the production of complex protein products and the limited production capacity of current systems. Addressing these will be integral in expanding the application of CFPS in biotherapy
Allen ME, Albon J, Elani Y, 2021, Layer-by-layer assembly of multi-layered droplet interface bilayers (multi-DIBs), Chemical Communications, Vol: 58, Pages: 60-63, ISSN: 1359-7345
Droplet interface bilayers (DIBs) have tremendous promise as platforms for fundamental biomembrane studies and in biotechnology. Being composed of a single bilayer however limits their biomimetic potential, as many cell membrane motifs are composed of multiple aligned bilayers. We describe a technology to manufacture cell-sized multi-layered DIBs (multi-DIBs) by coating giant unilamellar vesicles with a further monolayer, and allowing such structures to make contact with themselves or a monolayer coated droplet. This easily customisable strategy will pave the way for an expanded repertoire of DIB functionality, for example by facilitating the incorporation of multiple-bilayer spanning protein complexes.
Pazos MD, Hu Y, Elani Y, et al., 2021, Tattoo inks for optical biosensing in interstitial fluid, Advanced Healthcare Materials, Vol: 10, Pages: 1-22, ISSN: 2192-2640
The persistence of traditional tattoo inks presents an advantage for continuous andlong-term health monitoring in point of care devices. The replacement of tattoo pigments withoptical biosensors aims a promising alternative for monitoring blood biomarkers. Tattoo inksfunctionalization enables the control of interstitial biomarkers with correlated concentrations inplasma, to diagnose diseases, evaluate progression, and prevent complications associated withphysio pathological disorders or medication mismatches. The specific biomarkers in interstitialfluid provide a new source of information, especially for skin diseases. The study of tattoo inksdisplays insufficient regulation in their composition, a lack of reports of the relatedcomplications and a need for further studies on their degradation kinetics. This review focuseson tattoo optical biosensors for monitoring dermal interstitial biomarkers and discusses theyclinical advantages and main challenges for in vivo implantation. Tattoo functionalizationprovides a minimally invasive, reversible, biocompatible, real-time sensing with long-termpermanence and multiplexing capabilities for the control, diagnosis, and prevention of illness;it enables self-controlling management by the patient, but also the possibility of sending therecords to the doctor.
Lucey M, Ashik T, Marzook A, et al., 2021, Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking, Molecular Pharmacology, Vol: 100, Pages: 319-334, ISSN: 0026-895X
The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). Here we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and β-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency but exendin-4-C16 showed ~2.5-fold bias towards G protein recruitment and a ~60% reduction in β-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach, and a ~70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.
Ip T, Li Q, Brooks N, et al., 2021, Manufacture of multilayered artificial cell membranes through sequential bilayer deposition on emulsion templates., ChemBioChem: a European journal of chemical biology, Vol: 22, Pages: 2275-2281, ISSN: 1439-4227
Efforts to manufacture artificial cells that replicate the architectures, processes and behaviours of biological cells are rapidly increasing. Perhaps the most commonly reconstructed cellular structure is the membrane, through the use of unilamellar vesicles as models. However, many cellular membranes, including bacterial double membranes, nuclear envelopes, and organelle membranes, are multilamellar. Due to a lack of technologies available for their controlled construction, multilayered membranes are not part of the repertoire of cell-mimetic motifs used in bottom-up synthetic biology. To address this, we developed emulsion-based technologies that allow cell-sized multilayered vesicles to be produced layer-by-layer, with compositional control over each layer, thus enabling studies that would otherwise remain inaccessible. We discovered that bending rigidities scale with the number of layers and demonstrate inter-bilayer registration between coexisting liquid-liquid domains. These technologies will contribute to the exploitation of multilayered membrane structures, paving the way for incorporating protein complexes that span multiple bilayers.
Allen ME, Elani Y, Brooks NJ, et al., 2021, The effect of headgroup methylation on polymorphic phase behaviour in hydrated N-methylated phosphoethanolamine: palmitic acid membranes, Soft Matter, Vol: 17, Pages: 5763-5771, ISSN: 1744-683X
Mixtures of fatty acids and phospholipids can form hexagonal (HII) and inverse bicontinuous cubic phases, the latter of which are implicated in various cellular processes and have wide-ranging biotechnological applications in protein crystallisation and drug delivery systems. Therefore, it is vitally important to understand the formation conditions of inverse bicontinuous cubic phases and how their properties can be tuned. We have used differential scanning calorimetry and synchrotron-based small angle and wide angle X-ray scattering (SAXS/WAXS) to investigate the polymorphic phase behaviour of palmitic acid/ partially-methylated phospholipid mixtures, and how headgroup methylation impacts on inverse bicontinuous cubic phase formation. We find that upon partial methylation of the phospholipid headgroup (1 or 2 methyl substituents) inverse bicontinuous cubic phases are formed (of the Im3m spacegroup), which is not the case with 0 or 3 methyl substituents. This shows how important headgroup methylation is for controlling phase behaviour and how a change in headgroup methylation can be used to controllably tune various inverse bicontinuous phase features such as their lattice parameter and the temperature range of their stability.
Zhang S, Contini C, Hindley J, et al., 2021, Engineering motile aqueous phase-separated droplets via liposome stabilisation, Nature Communications, Vol: 12, Pages: 1-11, ISSN: 2041-1723
There are increasing efforts to engineer functional compartments that mimic cellular behaviours from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilised cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biological processes.
Elani Y, 2021, Interfacing living and synthetic cells as an emerging frontier in synthetic biology, Angewandte Chemie International Edition, Vol: 60, Pages: 5602-5611, ISSN: 1433-7851
The construction of artificial cells from inanimate molecular building blocks is one of the grand challenges of our time. In addition to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clinical and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re‐engineering living biological cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioural sophistication of their biological counterparts. Given this, many in the synthetic biology community have started to ask: is it possible to interface biological and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both? This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non‐living matter are blurred by bridging top‐down and bottom‐up synthetic biology. It details the state of play of this nascent field and introduces three generalised hybridisation modes that have emerged.
Pilkington CP, Seddon JM, Elani Y, 2021, Microfluidic technologies for the synthesis and manipulation of biomimetic membranous nano-assemblies., Physical Chemistry Chemical Physics, Vol: 23, Pages: 3693-3706, ISSN: 1463-9076
Microfluidics has been proposed as an attractive alternative to conventional bulk methods used in the generation of self-assembled biomimetic structures, particularly where there is a desire for more scalable production. The approach also allows for greater control over the self-assembly process, and parameters such as particle architecture, size, and composition can be finely tuned. Microfluidic techniques used in the generation of microscale assemblies (giant vesicles and higher-order multi-compartment assemblies) are fairly well established. These tend to rely on microdroplet templation, and the resulting structures have found use as comparmentalised motifs in artificial cells. Challenges in generating sub-micron droplets have meant that reconfiguring this approach to form nano-scale structures is not straightforward. This is beginning to change however, and recent technological advances have instigated the manufacture and manipulation of an increasingly diverse repertoire of biomimetic nano-assemblies, including liposomes, polymersomes, hybrid particles, multi-lamellar structures, cubosomes, hexosomes, nanodiscs, and virus-like particles. The following review will discuss these higher-order self-assembled nanostructures, including their biochemical and industrial applications, and techniques used in their production and analysis. We suggest ways in which existing technologies could be repurposed for the enhanced design, manufacture, and exploitation of these structures and discuss potential challenges and future research directions. By compiling recent advances in this area, it is hoped we will inspire future efforts toward establishing scalable microfluidic platforms for the generation of biomimetic nanoparticles of enhanced architectural and functional complexity.
Zhang S, Contini C, Hindley J, et al., 2020, Engineering motile aqueous phase-separated droplets via liposome stabilisation
<jats:title>Abstract</jats:title> <jats:p>There are increasing efforts to engineer functional compartments that mimic aspects of cellular behaviour in a drive to construct an artificial cell from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and the fact that movement of discrete cells is a ubiquitous feature of living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to issues with biocompatibility caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilized cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: if water is introduced into the emulsion system, these droplets can respond through directional motion away from PEG in the continuous phase and down to the polymer gradient with a velocity change proportional to the rearrangement of liposome stabilisers in the PEG/dextran interface. The biocompatibility, motility and partitioning abilities of this novel droplet system offers new directions to pursue research in motion-related biological processes.</jats:p>
Vivek A, Bolognesi G, Elani Y, 2020, Fusing artificial cell compartments and lipid domains using optical traps: a tool to modulate membrane composition and phase behaviour, Micromachines, Vol: 11, ISSN: 2072-666X
New technologies for manipulating biomembranes have vast potential to aid the understanding of biological phenomena, and as tools to sculpt novel artificial cell architectures for synthetic biology. The manipulation and fusion of vesicles using optical traps is amongst the most promising due to the level of spatiotemporal control it affords. Herein, we conduct a suite of feasibility studies to show the potential of optical trapping technologies to (i) modulate the lipid composition of a vesicle by delivering new membrane material through fusion events and (ii) manipulate and controllably fuse coexisting membrane domains for the first time. We also outline some noteworthy morphologies and transitions that the vesicle undergoes during fusion, which gives us insight into the mechanisms at play. These results will guide future exploitation of laser-assisted membrane manipulation methods and feed into a technology roadmap for this emerging technology.
Hindley JW, Zheleva DG, Elani Y, et al., 2019, Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells, Proceedings of the National Academy of Sciences, Vol: 116, Pages: 16711-16716, ISSN: 0027-8424
To date reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multi-compartment-spanning designer pathways that can be utilised to control downstream events inside an artificial cell, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.
Friddin MS, Elani Y, Trantidou T, et al., 2019, New directions for artificial cells using rapid prototyped biosystems, Analytical Chemistry, Vol: 91, Pages: 4921-4928, ISSN: 0003-2700
Microfluidics has been shown to be capable of generating a range of single- and multi- compartment vesicles and bilayer delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly recognized as powerful biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems and for screening drugs. One bottleneck in developing this technology is the time, expertise and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from Computer Aided Design (CAD) drawings. We highlight how this rapid prototyping revolution is transforming the fabrication of microfluidic devices for bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multi-billion dollar start-up could emerge from an attic or a basement.
Ces O, Elani Y, 2019, Community building in synthetic biology., Experimental biology and medicine (Maywood, N.J.), Vol: 244, Pages: 281-282, ISSN: 0037-9727
Friddin M, Bolognesi G, Salehi-Reyhani A, et al., 2019, Direct manipulation of liquid ordered lipid membrane domains using optical traps, Communications Chemistry, Vol: 2, Pages: 1-7, ISSN: 2399-3669
Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning.
Trantidou T, Dekker L, Polizzi K, et al., 2018, Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors, Interface Focus, Vol: 8, ISSN: 2042-8901
The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.
Trantidou T, Friddin M, Salehi-Reyhani S, et al., 2018, Droplet microfluidics for the construction of compartmentalised model membranes, Lab on a Chip, Vol: 18, Pages: 2488-2509, ISSN: 1473-0189
The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.
Bolognesi G, Friddin MS, Salehi-Reyhani S, et al., 2018, Sculpting and fusing biomimetic vesicle networks using optical tweezers, Nature Communications, Vol: 9, Pages: 1-11, ISSN: 2041-1723
Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.
Karamdad K, Hindley J, Friddin MS, et al., 2018, Engineering thermoresponsive phase separated vesicles formed via emulsion phase transfer as a content-release platform, Chemical Science, Vol: 9, Pages: 4851-4858, ISSN: 2041-6520
Giant unilamellar vesicles (GUVs) are a well-established tool for the study of membrane biophysics and are increasingly used as artificial cell models and functional units in biotechnology. This trend is driven by the development of emulsion-based generation methods such as Emulsion Phase Transfer (EPT), which facilitates the encapsulation of almost any water-soluble compounds (including biomolecules) regardless of size or charge, is compatible with droplet microfluidics, and allows GUVs with asymmetric bilayers to be assembled. However, the ability to control the composition of membranes formed via EPT remains an open question; this is key as composition gives rise to an array of biophysical phenomena which can be used to add functionality to membranes. Here, we evaluate the use of GUVs constructed via this method as a platform for phase behaviour studies and take advantage of composition-dependent features to engineer thermally-responsive GUVs. For the first time, we generate ternary GUVs (DOPC/DPPC/cholesterol) using EPT, and by compensating for the lower cholesterol incorporation efficiencies, show that these possess the full range of phase behaviour displayed by electroformed GUVs. As a demonstration of the fine control afforded by this approach, we demonstrate release of dye and peptide cargo when ternary GUVs are heated through the immiscibility transition temperature, and show that release temperature can be tuned by changing vesicle composition. We show that GUVs can be individually addressed and release triggered using a laser beam. Our findings validate EPT as a suitable method for generating phase separated vesicles and provide a valuable proof-of-concept for engineering content release functionality into individually addressable vesicles, which could have a host of applications in the development of smart synthetic biosystems.
Hindley JW, Elani Y, McGilvery CM, et al., 2018, Light-triggered enzymatic reactions in nested vesicle reactors, Nature Communications, Vol: 9, Pages: 1-6, ISSN: 2041-1723
Cell-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.
Elani 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: 2045-2322
There 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.
Thomas JM, Friddin MS, Ces O, et al., 2017, Programming membrane permeability using integrated membrane pores and blockers as molecular regulators, Chemical Communications, Vol: 53, Pages: 12282-12285, ISSN: 1359-7345
We report a bottom-up synthetic biology approach to engineering vesicles with programmable permeabilities. Exploiting the concentration-dependent relationship between constitutively active pores (alpha-hemolysin) and blockers allows blockers to behave as molecular regulators for tuning permeability, enabling us to systematically modulate cargo release kinetics without changing the lipid fabric of the system.
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