36 results found
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.
Strutt R, Sheffield F, Barlow N, et al., 2022, UV-DIB: label-free permeability determination using droplet interface bilayers, Lab on a Chip: miniaturisation for chemistry, physics, biology, materials science and bioengineering, Vol: 22, Pages: 972-985, ISSN: 1473-0189
Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis via ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low μM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industr
Kalossaka LM, Mohammed AA, Sena G, et al., 2021, 3D printing nanocomposite hydrogels with lattice vascular networks using stereolithography, JOURNAL OF MATERIALS RESEARCH, Vol: 36, Pages: 4249-4261, ISSN: 0884-2914
Kalossaka LM, Sena G, Barter LMC, et al., 2021, Review: 3D printing hydrogels for the fabrication of soilless cultivation substrates, Applied Materials Today, Vol: 24, Pages: 1-16, ISSN: 2352-9407
The use of hydrogels in academic research is fast evolving, and becoming more relevant to real life applications across varying fields. Additive Manufacturing (AM) has paved the way towards manufacturing hydrogel substrates with tailored properties which allow for new functionalities and applications. In this review, we introduce the idea of fabricating hydrogels as bioreceptive structures to be used as soilless cultivation substrates. AM is suggested as the fabrication process to achieve structures with features similar to soil. To evaluate this, we first review hydrogel fabrication processes, highlighting their key differences in terms of resolution, printing speed and build volume. Thus, we illustrate the examples from the literature where hydrogels were 3D printed with microorganisms such as algae. Finally, the challenges and future perspectives of printing soilless cultivation substrates are explored.
Kavanagh MA, Karlsson JK, Colburn JD, et al., 2020, A TDDFT investigation of the Photosystem II reaction center: Insights into the precursors to charge separation, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 19705-19712, ISSN: 0027-8424
Photosystem II (PS II) captures solar energy and directs charge separation (CS) across the thylakoid membrane during photosynthesis. The highly oxidizing, charge-separated state generated within its reaction center (RC) drives water oxidation. Spectroscopic studies on PS II RCs are difficult to interpret due to large spectral congestion, necessitating modeling to elucidate key spectral features. Herein, we present results from time-dependent density functional theory (TDDFT) calculations on the largest PS II RC model reported to date. This model explicitly includes six RC chromophores and both the chlorin phytol chains and the amino acid residues <6 Å from the pigments’ porphyrin ring centers. Comparing our wild-type model results with calculations on mutant D1-His-198-Ala and D2-His-197-Ala RCs, our simulated absorption-difference spectra reproduce experimentally observed shifts in known chlorophyll absorption bands, demonstrating the predictive capabilities of this model. We find that inclusion of both nearby residues and phytol chains is necessary to reproduce this behavior. Our calculations provide a unique opportunity to observe the molecular orbitals that contribute to the excited states that are precursors to CS. Strikingly, we observe two high oscillator strength, low-lying states, in which molecular orbitals are delocalized over ChlD1 and PheD1 as well as one weaker oscillator strength state with molecular orbitals delocalized over the P chlorophylls. Both these configurations are a match for previously identified exciton–charge transfer states (ChlD1+PheD1−)* and (PD2+PD1−)*. Our results demonstrate the power of TDDFT as a tool, for studies of natural photosynthesis, or indeed future studies of artificial photosynthetic complexes.
Haylock S, Friddin M, Hindley J, et al., 2020, Membrane protein mediated bilayer communication in networks of droplet interface bilayers, Communications Chemistry, Vol: 3, ISSN: 2399-3669
Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered ‘tissue-like’ networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.
Kavanagh M, Gould I, Barter L, 2019, Excited states of the photosystem II reaction center, ACS Fall National Meeting and Exposition, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
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.
Rains JGD, ODonnelly K, Oliver T, et al., 2019, Bicarbonate inhibition of carbonic anhydrase mimics hinders catalytic efficiency: Elucidating the mechanism and gaining insight toward improving speed and efficiency, ACS Catalysis, Vol: 9, Pages: 1353-1365, ISSN: 2155-5435
Carbonic anhydrase (CA) mimics are often studied with a focus on the hydration of CO2 for atmospheric carbon capture. Consequently, the reverse reaction (dehydration of HCO3–) has received minimal attention, so much so that the rate-limiting step of the dehydration reaction in CA mimics is currently unknown. The rate-limiting step of the hydration reaction is reported to be the bicarbonate-bound intermediate step, and thus is susceptible to product inhibition. It is not, however, clear if this inhibition is a consequence of an increase in the rate of the competing dehydration reaction or resulting from the strong affinity of bicarbonate to the mimic. To address this, insight into the dehydration reaction kinetics is needed. We therefore report the most comprehensive study of a CA mimic to date. The dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterized, and consequently evidence for the rate-limiting step for the dehydration reaction was seen to be the bicarbonate-bound intermediate step, much like the hydration reaction. This experimental validation of the rate-limiting step was achieved through a variety of methods including NMR experiments and the effect of inhibitors, substrate concentration, and metal center on activity. With this understanding, an improvement in the favorability of the rate-limiting step was achieved, resulting in decreased bicarbonate inhibition. Thus, an increase in the mimic’s kcat for both reactions was observed, resulting in the largest rate constants of any small-molecule CA mimic reported to date (28 093 and 579 M–1 s–1 for hydration and dehydration, respectively). Enzyme-like kcat/km values were obtained for ZnL1S (5.9 × 105 M–1 s–1 for CO2 hydration), and notably there is only a difference of 2.5 orders of magnitude from the enzyme, the closest of any CA mimic reported in the literature. The results from this work can be applied to the development and improvement
Barlow N, Kusumaatmaja H, Salehi-Reyhani A, et al., 2018, Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers, Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662
For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
Barlow NE, Bolognesi G, Haylock S, et al., 2017, Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress, Scientific Reports, Vol: 7, ISSN: 2045-2322
A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.
Sim S, Sowley H, Kidley N, et al., 2017, Investigation of inhibitor-protein interactions in plants & mammalians from EVV 2DIR data, 254th National Meeting and Exposition of the American-Chemical-Society (ACS) on Chemistry's Impact on the Global Economy, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Khanna T, Barter L, Gould I, 2017, Development and application of the AMBER molecular mechanics force field to investigate herbicide interaction in plants, 253rd National Meeting of the American-Chemical-Society (ACS) on Advanced Materials, Technologies, Systems, and Processes, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Barlow NE, Smpokou E, Friddin MS, et al., 2017, Engineering plant membranes using droplet interface bilayers, Biomicrofluidics, Vol: 11, ISSN: 1932-1058
Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.
We present a simple method for the multiplexed formation ofdroplet interface bilayers (DIBs) using a mechanically operatedlinear acrylic chamber array. To demonstrate the functionality ofthe chip design, a lipid membrane permeability assay is performed.We show that multiple, symmetric DIBs can be created andseparated using this robust low-cost approach.
Chan CL, Bolognesi G, Bhandarkar A, et al., 2016, DROPLAY: laser writing of functional patterns within biological microdroplet displays, Lab on a Chip, Vol: 16, Pages: 4621-4627, ISSN: 1473-0197
In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblieswith user-defined re-writable two-dimensional patterns of functional droplets. Light responsive water-in-oil dropletscapable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays in amicrofluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to ‘write’ userdefined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent)droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biologicalmonitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a largepopulation of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissuemimics and biological display materials.
Ces O, Elani Y, Karamdad K, et al., 2016, Novel microfluidic technologies for the bottom-up construction of artificial cells
This talk will outline novel microfluidic strategies for biomembrane engineering that are capable of fabricating vesicles , droplet interface bilayer networks , multisomes  and artificial tissues  where parameters such as membrane asymmetry, membrane curvature, compartment connectivity and individual compartment contents can be controlled. Various bulk methods, such as extrusion, gentle hydration and electroformation, have been synonymous with the formation of lipid vesicles over recent years. However these strategies suffer from significant shortcomings associated with these processes including limited control of vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. To address this technological bottleneck we have developed novel microfluidic platforms to form lipid vesicles in high-Throughput with full control over the composition of both the inner and outer leaflet of the membrane thereby enabling the manufacture of symmetric and asymmetric vesicles. This is achieved by manufacturing microfluidic channels with a step junction, produced by double-layer photolithography, which facilitates the transfer of a W/O emulsion across an oil-water phase boundary and the self-Assembly of a phospholipid bilayer. These platforms are being used to explore the role of asymmetry in biological systems  and study the engineering rules that regulate membrane mediated protein-protein interactions . In addition, these technologies are enabling the construction of biological machines capable of acting as micro-reactors , environmental sensors and smart delivery vehicles  as well as complex multi-compartment artificial cells where the contents and connectivity of each compartment can be controlled. These compartments are separated by biological functional membranes that can facilitate transport between the compartments themselves and between the compartments and external environment. This approach has led to the deve
O'Donnelly K, Zhao G, Patel P, et al., 2014, Isolation and kinetic characterisation of hydrophobically distinct populations of form I Rubisco, Planet Methods, Vol: 10, ISSN: 1746-4811
BackgroundRubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco’s activity could have a significant impact on photosynthetic efficiency and crop yield.ResultsThis paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity.ConclusionsWe demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco’s structural and functional properties.
Miller D, Booth PJ, Seddon JM, et al., 2013, Protocell design through modular compartmentalization, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 10, ISSN: 1742-5689
Wormit A, Butt SM, Chairam I, et al., 2012, Osmosensitive Changes of Carbohydrate Metabolism in Response to Cellulose Biosynthesis Inhibition, PLANT PHYSIOLOGY, Vol: 159, Pages: 105-117, ISSN: 0032-0889
Charalambous K, Booth PJ, Woscholski R, et al., 2012, Engineering <i>de Novo</i> Membrane-Mediated Protein-Protein Communication Networks, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 134, Pages: 5746-5749, ISSN: 0002-7863
Fournier F, Gardner EM, Guo R, et al., 2008, Optical fingerprinting of peptides using two-dimensional infrared spectroscopy: Proof of principle, ANALYTICAL BIOCHEMISTRY, Vol: 374, Pages: 358-365, ISSN: 0003-2697
Donaldson PM, Guo R, Fournier F, et al., 2007, Direct identification and decongestion of Fermi resonances by control of pulse time ordering in two-dimensional IR spectroscopy (vol 127, art no. 114513, 2007), JOURNAL OF CHEMICAL PHYSICS, Vol: 127, ISSN: 0021-9606
Barter LMC, Klug DR, Woscholski R, 2007, Does history repeat itself? The emergence of a new discipline (vol 1, pg 737, 2006), ACS CHEMICAL BIOLOGY, Vol: 2, Pages: 271-271, ISSN: 1554-8929
Donaldson PM, Guo R, Fournier F, et al., 2007, Direct identification and decongestion of Fermi Resonances by control of pulse time-ordering in 2D-IR Spectroscopy, Journal Of Chemical Physics
Barter LMC, Klug DR, Woscholski R, 2006, Does history repeat itself? The emergence of a new discipline, ACS CHEMICAL BIOLOGY, Vol: 1, Pages: 737-740, ISSN: 1554-8929
Barter LMC, Klug DR, 2005, A unified picture of energy and electron transfer in primary photosynthesis, CHEMICAL PHYSICS, Vol: 319, Pages: 308-315, ISSN: 0301-0104
Barter LMC, van Grondelle R, Klug DR, 2005, Energy trapping and equilibration: a balance of regulation and efficiency, Photosystem II: the light-driven water: plastoquinone oxidoreductase, Editors: Wydrzynski, Satoh, Wydrzynski, Satoh, Publisher: Springer, Pages: 491-514, ISBN: 9781402042492
Barter LMC, Durrant JR, Klug DR, 2003, A quantitative structure-function relationship for the Photosystern II reaction center: Supermolecular behavior in natural photosynthesis, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 100, Pages: 946-951, ISSN: 0027-8424
Barter LMC, Schilstra MJ, Barber J, et al., 2001, Are the trapping dynamics in Photosystem II sensitive to Q<sub>A</sub> redox potential?, JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY, Vol: 142, Pages: 127-132, ISSN: 1010-6030
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