In the discipline of bottom-up synthetic biology, vesicles define boundaries artificial cells and are increasingly being used as biochemical microreactors operating in physiological environments. As field matures, there is a need to compartmentalize processes different spatial localities within vesicles, for these interact with one another. Here we address this by designing constructing multi-compartment which an engineered multi-step enzymatic pathway carried out. The individual steps...

Polydimethylsiloxane (PDMS) is a dominant material in the fabrication of microfluidic devices to generate water-in-oil droplets, particularly lipid-stabilized because its highly hydrophobic nature. However, key property hydrophobicity has hindered use generation oil-in-water which requires channels have hydrophilic surface properties. In this article, we developed, optimized, and characterized method produce PDMS with via deposition polyvinyl alcohol following plasma treatment demonstrated...

Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- nanocompartments are used for performing host highly specialized tightly regulated functions. The benefit compartmentalization as design principle behind its ubiquity cells has led to it being central engineering theme construction artificial cell-like systems. In this review, we discuss the attractions designing compartmentalized membrane-bound constructs review...

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge their physical state and structure. However, mapping viscosity diffusion in complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy imaging (FLIM) meso-substituted BODIPY molecular rotor localised leaflet model various compositions. prepare large giant unilamellar vesicles (LUVs GUVs) containing phosphatidylcholine (PC) lipids...

In recent years it has become evident that many biological functions and processes are associated with the adoption by cellular membranes of complex geometries, at least locally. this paper, we initially discuss range self-assembled structures lipids, building blocks membranes, may form, focusing specifically on inverse lyotropic phases negative interfacial mean curvature. We describe roles curvature elasticity packing frustration in controlling stability these phases, experimental...

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction inverse cubic phases phase transitions in lipids. §1 , briefly discuss the lyotropic behaviour lipids, focusing attention on non-lamellar structures, their geometric/topological relationship to fusion processes lipid membranes. Possible pathways for between different are also outlined. §2 effects hydrostatic pressure membranes transitions, describe how parameters required predict dependence...

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This is ultimately derived the ability compartmentalise and order chemical species space. To unlock such applications, spatial organisation of vesicles relation one another must be controlled, techniques deliver cargo compartments developed. Herein, we use optical tweezers assemble, reconfigure dismantle cell-sized that,...

Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology chassis for artificial cells. In these areas the introduction of light-responsive modules affords increased functionality, example, to initiate enzymatic reactions vesicle interior with spatiotemporal control. Here we report a system composed nested where inner compartments act phototransducers, responding ultraviolet irradiation through diacetylene polymerisation-induced...

To date, reconstitution of one the fundamental methods cell communication, signaling pathway, has been unaddressed in bottom-up construction artificial cells (ACs). Such developments are needed to increase functionality and biomimicry ACs, accelerating their translation application biotechnology. Here, we report a de novo synthetic pathway microscale nested vesicles. Vesicle-cell models respond external calcium signals through activation an intracellular interaction between phospholipase A2...

Whereas spatial organisation of function is ubiquitous in biology, it has been lacking artificial cells. We rectify this by using multi-compartment vesicles as chassis for cells, allowing distinct biological processes to be isolated space. This demonstrated vitro synthesis two proteins predefined vesicle regions.

We have constructed swollen bicontinuous cubic lipid phases from monoglyceride, anionic and cholesterol. These self-assembled systems lattice parameters of almost 50 nm, over 4 times larger than archetypal phases.

We detail an approach for constructing asymmetric membranes and characterising their mechanical properties, leading to the first measurement of effect asymmetry on lipid bilayer mechanics.

There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity lack of evolved biochemical pathways, the capabilities are limited comparison counterparts. We show that encapsulating living provides a means for leverage cellular biochemistry, encapsulated serving organelle-like functions as modules inside larger cell assembly. Using microfluidic technologies construct such hybrid bionic...

We present a novel method for the generation of giant unilamellar vesicles and use fluctuation analysis to characterise their mechanical properties.

Abstract The rapid development of nanotechnology has led to an increase in the number and variety engineered nanomaterials environment. Gold nanoparticles (AuNPs) are example a commonly studied nanomaterial whose highly tailorable properties have generated significant interest through wide range research fields. In present work, we characterise AuNP-lipid membrane interaction by coupling qualitative data with quantitative measurements enthalpy change interaction. We investigate interactions...

The liquid crystalline lamellar (${L}_{\ensuremath{\alpha}}$) to double-diamond inverse bicontinuous cubic (${Q}_{\mathrm{II}}^{D}$) phase transition for the amphiphile monoelaidin in excess water exhibits a remarkable sequence of structural transformations pressure or temperature jumps. Our data imply that dynamics depends on coupling between changes molecular shape and geometrical topological constraints domain size. We propose qualitative model this based theories membrane fusion via...

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby cells are manipulated into separate chambers using an optical trap; lysed by shock wave caused laser-induced microcavitation, and released cell measured total internal reflection microscopy as it bound micro-printed antibody spots within device. was tested GFP transfected relative precision measurement method determined be 88%. Single...

Many amphiphile–water mixtures will self-assemble into three-dimensional soft condensed structures known as inverse bicontinuous cubic phases. These are found in nature and have applications nanotechnology. Here we show that by systematically varying amphiphile chain splay, able to control the relative stability of phases a homologous series monoglycerides predictable manner. In particular, demonstrate decreasing splay leads appearance primitive phase while increasing reduces channel size...

Vesicles serve important functions in the construction of artificial cells. They facilitate biochemical reactions by confining reactants and products space, delineate boundaries protocell. allow concentration gradients to form, control passage molecules via embedded proteins. However, date, manufacturing strategies have focussed on uni-compartmental structures, resulting vesicles with homogenous internal content. This is contrast real cells which spatial segregation components processes. We...

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting plastids 1 (Vipp1), functions maintenance thylakoid biogenesis cyanobacteria plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices required binding; however, the features recognized expressing their functionalities have remained largely uncharacterized. Rigorously controlled, vitro methodologies...

Bicontinuous cubic structures offer enormous potential in applications ranging from protein crystallisation to drug delivery systems and have been observed cellular membrane structures. One of the current bottlenecks understanding exploiting these is that scaffolds produced vitro are considerably smaller size than those biological systems, differing by almost an order magnitude some cases. We addressed this technological bottleneck developed a methodology capable manufacturing highly swollen...