DIB SCREENING · Rapid functional characterization of ion channels with droplet interface bilayers

Rapid functional characterization of ion channels with droplet interface bilayers

Project context and objectives The goal of my proposed work was to develop a rapid screening platform for determining the functional activity of ion channels in the presence of pharmacological molecules such as ion channel blockers. My specific aim was to screen ion channel libraries against a variety of blockers and thereby localise the interaction sites. Such an assay should be a fast, cost-effective, information-rich method, employing parallel or quickly reproducible processing steps. The development of this assay was divided into three stages: - the design and implementation of an automated rapid screening platform based on a droplet interface bilayer (DIB); - the development of protocols for entrapping single deoxyribonucleic acid (DNA) plasmids in lipid monolayer-encased aqueous droplets for coupled in vitro transcription-translation (IVTT); - the automation of microdroplet generation by using microfluidic systems, and combining the modules to obtain a functional set-up. Work performed Lipid-coated aqueous droplets can be filled with the essential cellular protein machinery to synthesise membrane proteins or pores. These droplet 'protocells' are able to communicate through interface bilayers thus making them promising components of synthetic minimal tissues. We have shown that aqueous droplets in oil can be connected by lipid bilayers through which they can communicate by means of ion channels and pores. Two-dimensional (2-D) droplet networks have been constructed, which function as light sensors, batteries and simple electronic circuits. We have reported the manual assembly of lipid-coated aqueous droplets in oil to form 2-D networks and the manual encapsulation of simple droplet networks so that they can function in an aqueous environment. A principal goal of synthetic biology is to reprogram cells or build them from scratch. Work on reprogramming was focused on reconstructed and synthetic genomes. An alternative, bottom-up approach is to build cells from parts, with the goals of mimicking and complementing the properties of biological cells, and even understanding the origin of life. These synthetic minimal cells, or protocells, are expected to perform various actions, such as energy storage and utilisation, linear and rotary motion, sensing and signal transduction, the uptake, transformation and release of small molecules, and computation. Far less work has been done on assemblies of minimal cells, minimal tissues or prototissues, which might be better able to carry out these tasks and also to produce emergent properties that cannot be achieved by individual cells. For example, by placing protein pores in the bilayers between constituent droplets, we defined electrical and chemical communication pathways in a 14-droplet 3-D pyramidal assembly. Our experiments demonstrate the construction of designed 3-D droplet networks in which each droplet can contain different proteins or small molecules. The assemblies are functional units capable of internal and external communication. A key advance towards the fabrication of more intricate synthetic tissues would be the ability to construct 3-D droplet networks in a precisely controlled manner. Here, we have developed an improved means of droplet assembly that utilises magnetic manipulation. The ability to pick and place droplets in 3-D structures is an important step towards the programmed and automated manufacture of synthetic minimal tissues (prototissues). Simple droplet networks assembled with the magnetic technique were used as construction modules to form more complex functional 2-D and 3-D structures. Our ongoing efforts are focused on building functional synthetic minimal tissues by using 3-D networks of droplets containing different molecules to perform specific functions, akin to the differentiated cells of biological tissues. The combination of different functional modules will allow the formation of minimal tissues that accept inputs, process them and give a variety of outputs. These materials may find applications in medicine because they can be controlled electronically, with light or with a magnetic field. We speculate that 3-D droplet networks might be integrated with living tissues to repair them or enhance their properties. Two and three-dimensional aqueous droplet networks are delicate. We envisage that for biotechnological applications, and even for gaining a basic mechanistic understanding of membrane proteins, a new robust bilayer platform amenable to manipulation is needed. Such a robust 3-D bilayer system would be able to incorporate engineered membrane proteins for inter-compartment communication, which could be studied with optical and electrical techniques. Hydrogels are ideal scaffolds to realise such systems: they conduct ions, they can be moulded and many are biocompatible. Main results We used a simple approach in which shaped lipid-coated hydrogel objects were used to assemble networks, both with and without bilayers between them. A membrane pore such as a-hemolysin could be inserted when the interfacial bilayer was formed. We further showed that lipid-coated hydrogel shapes can be used as building blocks for networks, yielding scalable electrical circuits and mechanical devices. Examples include a mechanical switch, a rotor driven by a magnetic field and painted circuits, analogous to printed circuit boards, made with centimetre-length agarose wires. Bottom-up fabrication with lipid-coated hydrogel shapes is therefore a useful step towards the synthetic biology of functional devices including minimal tissues. Socioeconomic impact of the project The work initiated here might be extended in several directions. The assembly of aqueous droplets or hydrogel objects might be controlled by shape, surface energy or molecular recognition. Temperature, pH, light, chemicals, ions, and magnetic or electrical fields have been shown to act as stimuli for switching the shapes of hydrogels and might therefore be used to control assembly, as might surfaces with switchable properties. One long-term goal is the fabrication of biocompatible synthetic tissues for the delivery of therapeutic agents. Such synthetic tissues would have to be adapted to work in an aqueous environment, which has already been achieved with droplet networks. The synthetic tissues might be autonomous or operate through external impetuses, such as light or a magnetic field. Alternatively, synthetic tissues might be connected to electronic interfaces. Again, a direct interface between natural and synthetic tissues might be made, in which case the shape of the hydrogel compartments in contact with natural cells would be all-important. The ability to change shape, by further analogy with the cytoskeleton, might also be useful in this regard.

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