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      Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli

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          Abstract

          Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid–protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.

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          Droplet interface bilayers.

          Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.
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            Droplet networks with incorporated protein diodes show collective properties.

            Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.
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              Novel technologies for the formation of 2-D and 3-D droplet interface bilayer networks.

              Droplet interface bilayer (DIB) networks have vast potential in the field of membrane biophysics, synthetic biology, and functional bio-electronics. However a technological bottleneck exists in network fabrication: existing methods are limited in terms of their automation, throughput, versatility, and ability to form well-defined 3-D networks. We have developed a series of novel and low-cost methodologies which address these limitations. The first involves building DIB networks around the contours of a microfluidic chip. The second uses flow rate and droplet size control to influence droplet packing geometries within a microfluidic chamber. The latter method enables the controlled formation of various 3-D network arrays consisting of thousands of interconnected symmetric and asymmetric lipid bilayers for the first time. Both approaches allow individual droplet position and composition to be controlled, paving the way for complex on-chip functional network synthesis.
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                Author and article information

                Journal
                J R Soc Interface
                J R Soc Interface
                RSIF
                royinterface
                Journal of the Royal Society Interface
                The Royal Society
                1742-5689
                1742-5662
                6 September 2014
                6 September 2014
                : 11
                : 98
                : 20140404
                Affiliations
                [1 ]Membrane Biophysics Platform, Institute of Chemical Biology and Department of Chemistry, Imperial College London , South Kensington, London SW7 2AX, UK
                [2 ]Department of Biochemistry, University of Bristol , Bristol BS8 1TD, UK
                [3 ]Pfizer Global Research and Development , Sandwich CT13 9NJ, UK
                Author notes
                Article
                rsif20140404
                10.1098/rsif.2014.0404
                4233688
                25008079
                9cf5ab9f-e7b0-4566-a5be-16a79e702d63

                © 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.

                History
                : 17 April 2014
                : 13 June 2014
                Categories
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                September 6, 2014

                Life sciences
                droplet interface bilayers,mechanosensitive channel of large conductance,1,2-dioleoyl-sn-glycero-3-phosphocholine,fluorescence

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