Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers

Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) PEG 8000 and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can therefore be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channel

Programming membrane permeability using integrated membrane pores and blockers as molecular regulators

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

Towards a skin-on-a-chip for screening the dermal absorption of cosmetics

Over the past few decades, there have been increasing global efforts to limit or ban the use of animals for testing cosmetic products. This ambition has been at the heart of international endeavours to develop new in vitro and animal-free approaches for assessing the safety of cosmetics. While several of these new approach methodologies (NAMs) have been approved for assessing different toxicological endpoints in the UK and across the EU, there remains an absence of animal-free methods for screening for dermal absorption; a measure that assesses the degree to which chemical substances can become systemically available through contact with human skin. Here, we identify some of the major technical barriers that have impacted regulatory recognition of an in vitro skin model for this purpose and propose how these could be overcome on-chip using artificial cells engineered from the bottom-up. As part of our future perspective, we suggest how this could be realised using a digital biomanufacturing pipeline that connects the design, microfluidic generation and 3D printing of artificial cells into user-crafted synthetic tissues. We highlight milestone achievements towards this goal, identify future challenges, and suggest how the ability to engineer animal-free skin models could have significant long-term consequences for dermal absorption screening, as well as for other applications

Mask-free laser lithography for rapid and low-cost microfluidic device fabrication

Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography—a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 μm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics

A transparent 3D printed device for assembling droplet hydrogel bilayers (DHBs)

We report a new approach for assembling droplet hydrogel bilayers (DHBs) using a transparent 3D printed device. We characterise the transparency of our platform, confirm bilayer formation using electrical measurements and show that single-channel recordings can be obtained using our reusable rapid prototyped device. This method significantly reduces the cost and infrastructure required to develop devices for DHB assembly and downstream study

Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets

We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems

Direct manipulation of liquid ordered lipid membrane domains using optical traps

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

Mask-free laser lithography for rapid and low-cost microfluidic device fabrication

Copyright © 2018 American Chemical Society. Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography - a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 μm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics

Sculpting and fusing biomimetic vesicle networks using optical tweezers

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

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology. In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a ‘millifluidic’ chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7–6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2–9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm2, or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24–48 hours