In the discipline of bottom-up synthetic biology, vesicles define the boundaries of artificial cells and are increasingly being used as biochemical microreactors operating in physiological environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymatic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chemical pathway to be separated in space, this platform bridges the gap between table-top chemistry and chemistry that is performed within vesicles.

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. In this paper, we initially discuss the range of self-assembled structures that lipids, the building blocks of biological membranes, may form, focusing specifically on the inverse lyotropic phases of negative interfacial mean curvature. We describe the roles of curvature elasticity and packing frustration in controlling the stability of these inverse phases, and the experimental determination of the spontaneous curvature and the curvature elastic parameters. We discuss how the lyotropic phase behaviour can be tuned by the addition of compounds such as long-chain alkanes, which can relieve packing frustration. The latter section of the paper elaborates further on the structure, geometric properties, and stability of the inverse bicontinuous cubic phases.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Polydimethylsiloxane (PDMS) is a dominant material in the fabrication of microfluidic devices to generate water-in-oil droplets, particularly lipid-stabilized droplets, because of its highly hydrophobic nature. However, its key property of hydrophobicity has hindered its use in the microfluidic generation of oil-in-water droplets, which requires channels to have hydrophilic surface properties. In this article, we developed, optimized, and characterized a method to produce PDMS with a hydrophilic surface via the deposition of polyvinyl alcohol following plasma treatment and demonstrated its suitability for droplet generation. The proposed method is simple, quick, effective, and low cost and is versatile with respect to surfactants, with droplets being successfully generated using both anionic surfactants and more biologically relevant phospholipids. This method also allows the device to be selectively patterned with both hydrophilic and hydrophobic regions, leading to the generation of double emulsions and inverted double emulsions.

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In [section sign] 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In [section sign] 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In [section sign] 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In [section sign] 4, we describe an IDL-based 'AXcess' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In [section sign] 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in [section sign] 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.