Research reported in this publication is supported by the Air Force Office of Scientific Research Basic Initiative Grant FA9550-12-1-0464.

<ol>
	<li>Montal, M., Mueller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bimolecular+membranes+from+lipid+monolayers+and+a+study+of+their+electrical+properties.">Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties.</a> P NATL ACAD SCI USA. 69, 3561-3566 (1972).</li><li>Tsofina, L., Liberman, E., Babakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+bimolecular+protein-lipid+membranes+in+aqueous+solution.">Production of bimolecular protein-lipid membranes in aqueous solution.</a> Nature. , (1966).</li><li>Funakoshi, K., Suzuki, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Bilayer+Formation+by+Contacting+Monolayers+in+a+Microfluidic+Device+for+Membrane+Protein+Analysis.">Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis.</a> ANAL CHEM. 78, 8169-8174 (2006).</li><li>Holden, M. A., Needham, D., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+bionetworks+from+nanoliter+water+droplets.">Functional bionetworks from nanoliter water droplets.</a> J AM CHEM SOC. 129, 8650-8655 (2007).</li><li>Hwang, W. L., Chen, M., Cronin, B., Holden, M. A., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+droplet+interface+bilayers.">Asymmetric droplet interface bilayers.</a> J AM CHEM SOC. 130, 5878-5879 (2008).</li><li>Maglia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+networks+with+incorporated+protein+diodes+show+collective+properties.">Droplet networks with incorporated protein diodes show collective properties.</a> Nat Nano. 4, 437-440 (2009).</li><li>Sarles, S. A., Stiltner, L. J., Williams, C. B., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayer+formation+between+lipid-encased+hydrogels+contained+in+solid+substrates.">Bilayer formation between lipid-encased hydrogels contained in solid substrates.</a> ACS APPL MATER INTER. 2, 3654-3663 (2010).</li><li>Sarles, S. A., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+attachment+method+for+reconstituting+lipid+bilayers+of+prescribed+size+within+flexible+substrates.">Regulated attachment method for reconstituting lipid bilayers of prescribed size within flexible substrates.</a> ANAL CHEM. 82, 959-966 (2010).</li><li>Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical Encapsulation of Interface Bilayers. , (2010).</li><li>Sarles, S. A., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-based+biomolecular+smart+materials.">Membrane-based biomolecular smart materials.</a> SMART MATER STRUCT. 20, 094018 (2011).</li><li>Sarles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+virtual+ground+to+control+transmembrane+voltages+and+measure+bilayer+currents+in+serial+arrays+of+droplet+interface+bilayers.">The use of virtual ground to control transmembrane voltages and measure bilayer currents in serial arrays of droplet interface bilayers.</a> SMART MATER STRUCT. 22, 094023 (2013).</li><li>Sarles, S. A., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-inspired+electroactive+polymer+materials+incorporating+biomolecular+materials.">Cell-inspired electroactive polymer materials incorporating biomolecular materials.</a> SPIE Smart Structures and Materials+ Nondestructive Evaluation and Health Monitoring. , 797626 (2011).</li><li>Hwang, W. L., Chen, M., Cronin, B., Holden, M. A., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+droplet+interface+bilayers.">Asymmetric droplet interface bilayers.</a> Journal of the American Chemical Society. 130, 5878-5879 (2008).</li><li>Bayley, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+interface+bilayers.">Droplet interface bilayers.</a> MOL BIOSYST. 4, 1191-1208 (2008).</li><li>White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+torus+surrounding+planar+lipid+bilayer+membranes.">Analysis of the torus surrounding planar lipid bilayer membranes.</a> BIOPHYS J. 12, 432 (1972).</li><li>Tien, H. T., Ottova, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+bilayer+concept+and+its+experimental+realization:+from+soap+bubbles,+kitchen+sink,+to+bilayer+lipid+membranes.">The lipid bilayer concept and its experimental realization: from soap bubbles, kitchen sink, to bilayer lipid membranes.</a> J MEMBRANE SCI. 189, 83-117 (2001).</li><li>Perozo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+prokaryotic+mechanosensitive+channels.">Gating prokaryotic mechanosensitive channels.</a> NAT REV MOL CELL BIO. 7, 109-119 (2006).</li><li>Kung, C., Martinac, B., Sukharev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+channels+in+microbes.">Mechanosensitive channels in microbes.</a> ANNU REV MICROBIOL. 64, 313-329 (2010).</li><li>Bialecka-Fornal, M., Lee, H. J., DeBerg, H. A., Gandhi, C. S., Phillips, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+census+of+mechanosensitive+channels+in+living+bacteria.">Single-cell census of mechanosensitive channels in living bacteria.</a> PLoS ONE. 7, e33077 (2012).</li><li>Levina, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+Escherichia+coli+cells+against+extreme+turgor+by+activation+of+MscS+and+MscL+mechanosensitive+channels:+identification+of+genes+required+for+MscS+activity.">Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity.</a> The EMBO Journal. 18, 1730-1737 (1999).</li><li>Chiang, C. -. S., Anishkin, A., Sukharev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+of+the+large+mechanosensitive+channel+in+situ:+estimation+of+the+spatial+scale+of+the+transition+from+channel+population+responses.">Gating of the large mechanosensitive channel in situ: estimation of the spatial scale of the transition from channel population responses.</a> BIOPHYS J. 86, 2846-2861 (2004).</li><li>Anishkin, A., Chiang, C. -. S., Sukharev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+mutations+reveal+expanded+intermediate+states+and+a+sequential+action+of+two+gates+in+MscL.">Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL.</a> J GEN PHYSIOL. 125, 155-170 (2005).</li><li>Sukharev, S. I., Sigurdson, W. J., Kung, C., Sachs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+and+Spatial+Parameters+for+Gating+of+the+Bacterial+Large+Conductance+Mechanosensitive+Channel,+MscL.">Energetic and Spatial Parameters for Gating of the Bacterial Large Conductance Mechanosensitive Channel, MscL.</a> J GEN PHYSIOL. 113, 525-540 (1999).</li><li>Deplazes, E., Louhivuori, M., Jayatilaka, D., Marrink, S. J., Corry, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+investigation+of+MscL+gating+using+experimental+data+and+coarse+grained+MD+simulations.">Structural investigation of MscL gating using experimental data and coarse grained MD simulations.</a> PLOS COMPUT BIOL. 8, e1002683 (2012).</li><li>Kubalski, A., Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial ion channels and their eukaryotic homologs. , (2005).</li><li>Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+MscL+homolog+from+Mycobacterium+tuberculosis:+a+gated+mechanosensitive+ion+channel.">Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel.</a> Science. 282, 2220-2226 (1998).</li><li>Steinbacher, S., Bass, R., Strop, P., Rees, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+prokaryotic+mechanosensitive+channels+MscL+and+MscS.">Structures of the prokaryotic mechanosensitive channels MscL and MscS.</a> Mechanosensitive Ion Channels, Part A. , 1-24 (2007).</li><li>Sukharev, S., Durell, S. R., Guy, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+models+of+the+MscL+gating+mechanism.">Structural models of the MscL gating mechanism.</a> BIOPHYS J. 81, 917-936 (2001).</li><li>Perozo, E., Cortes, D. M., Sompornpisut, P., Kloda, A., Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+channel+structure+of+MscL+and+the+gating+mechanism+of+mechanosensitive+channels.">Open channel structure of MscL and the gating mechanism of mechanosensitive channels.</a> Nature. 418, 942-948 (2002).</li><li>Betanzos, M., Chiang, C. -. S., Guy, H. R., Sukharev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+iris-like+expansion+of+a+mechanosensitive+channel+protein+induced+by+membrane+tension.">A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension.</a> NAT STRUCT MOL BIOL. 9, 704-710 (2002).</li><li>Corry, B., Jayatilaka, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulation+of+structure,+orientation,+and+energy+transfer+between+AlexaFluor+molecules+attached+to+MscL.">Simulation of structure, orientation, and energy transfer between AlexaFluor molecules attached to MscL.</a> BIOPHYS J. 95, 2711-2721 (2008).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+FRET+Reveals+Pore+Size+and+Opening+Mechanism+of+MscL.">Single Molecule FRET Reveals Pore Size and Opening Mechanism of MscL.</a> eLife. 3, e01834 (2014).</li><li>Anishkin, A., Akitake, B., Kamaraju, K., Chiang, C., Sukharev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydration+properties+of+mechanosensitive+channel+pores+define+the+energetics+of+gating.">Hydration properties of mechanosensitive channel pores define the energetics of gating.</a> J Phys Condens Matter. 22, 454120 (2010).</li><li>Ko&#231;er, A., Walko, M., Meijberg, W., Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-actuated+nanovalve+derived+from+a+channel+protein.">A light-actuated nanovalve derived from a channel protein.</a> Science. 309, 755-758 (2005).</li><li>Najem, J., Dunlap, M., Sukharev, S., Leo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+Channels+Activity+in+a+Droplet+Interface+Bilayer+System.">Mechanosensitive Channels Activity in a Droplet Interface Bilayer System.</a> MRS Proceedings. 1621, (2014).</li><li>Fox, R. O., Richards, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+voltage-gated+ion+channel+model+inferred+from+the+crystal+structure+of+alamethicin+at+1.5-&#8491;+resolution.">A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-&#8491; resolution.</a> Nature. 300, 325-330 (1982).</li><li>Iwamoto, M., Oiki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+Bubble+Bilayers+with+Flush+Drainage.">Contact Bubble Bilayers with Flush Drainage.</a> Sci Rep. 5, (2015).</li><li>Barriga, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+interface+bilayer+reconstitution+and+activity+measurement+of+the+mechanosensitive+channel+of+large+conductance+from+Escherichia+coli.">Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.</a> J R Soc Interface. 11, 20140404 (2014).</li></ol>

The authors have nothing to disclose.

Mechanosensation signifies one of the first sensory transduction pathways that evolved in living organisms. Using this phenomenon for studying and understanding the mechano-electrical properties of the DIB, is a crucial step toward functional stimuli-responsive materials. It involves the incorporation and activation of a mechanosensitive channel, MscL, in the DIB as a mechanoelectrical transducer and a strain gauge to detect tension increase in the lipid bilayer interface. On another note, the function of MS channels could be regulated through the basic material properties of lipid bilayers including thickness, intrinsic curvature, and compressibility. In light of the aforementioned, the micropipette-based technique provides a valuable tool allowing the researcher the ability to study MS channels in DIBs and provides insights into the structure of the lipid bilayer, as well as the lipid-protein interactions.
Over the past three decades, patch-clamp was the primary method to study MS channels, since it allows clamping of both voltage and tension. However, patch-clamp requires bulky equipment and not suitable for miniaturization, a property required for the engineering of sensory and conversion devices. DIBs due to their simplicity, stability, and compactness represent a suitable environment to study the activity of MscL. Here, we extend previous advances in the DIB formation techniques by proposing a micropipette-based technique, with the ability to control the size of droplets and bilayer interface, the chemical composition of each droplet, and the tension at the interface through dynamic stimulation. The technique consists of anchoring aqueous droplets, containing proteoliposomes, to the tips of coaxially opposing glass capillaries. The droplets are placed in a bath of organic solvent and when brought in contact a lipid bilayer forms at the interface.
The micropipettes are attached to piezoelectric oscillators, allowing horizontal displacement of the droplets. Dynamically compressing the droplets, results in an increase of interfacial tension at the water oil interface and therefore an increase in bilayer tension. Two major aspects differentiate this method from the similar and recently published contact bubble bilayer (CBB) technique37. Using the technique presented herein, the size of the bilayer is controlled using micromanipulators and thus the volumes of the droplets remain constant, unlike in the CBB method. In addition, the CBB technique calls for pressure pumps, which are not needed in the method presented in this paper making it simpler and easier to build.
We are able to incorporate and stimulate bacterial MscL for the first time without the use of a patch pipette or chemical modifications38. Since the system facilitates the formation of robust asymmetric lipid bilayer membranes, it more closely mimics the lipid asymmetry found in biological membranes. This allows us to study the effects of controlled membrane composition or asymmetry on the activity of MscL. Additionally, through image processing techniques, this method helps estimate the tension at the bilayer interface. This technique assists in understanding the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves the understanding of MscL response to membrane tension.
Although this method takes us a step closer toward a biomolecular stimuli-responsive material system and to a different physiological environment to study MscL, there are limitations to the system. Tension in this system cannot be clamped due to the presence of the lipid reservoir in the form of liposomes in each droplet, which tends to relieve tension at the oil/water interface. Therefore, at present mechanosensitive channels can be stimulated in DIBs only in a dynamic regime. The presence of air bubbles in the system significantly affects the precision and reproducibility of the experiments. Air bubbles present in the hydrogels could result loss if electrical connection.
While we describe the use of the micro-pipette based method for the stimulation of MscL, the technique could be used to study other types of MS channels and has the potential to be used by researchers to study a variety of biomolecules. For instance, similar setup has been used in our lab to study the mechanoelectrical response of a channel-free droplet interface bilayer membrane. Various proteins could be reconstituted and activated using this highly controlled setup, taking in consideration that the reconstitution environments of each biomolecule vary. The method described in this article touches on a considerably wider application potential that is only limited to the imagination of the researcher.

In the past decade, the assembly of artificial lipid bilayers has been substantially advanced through the development of the droplet interface bilayer method. Known as stable and robust, DIBs imposed themselves as alternative model systems to the classical painted (Mueller) and folded (Montal-Mueller) planar bilayers1. Although the idea of using droplets to create lipid bilayers dates back to the 1960s2, it has not gained popularity until recently. The first successful attempt was reported by the Takeushi group3, followed by several studies demonstrating bilayer formation using a network of droplets by the Bayley group4-6. More recently, encapsulation techniques were proposed by the Leo group7-9, who pioneered the concept of using DIBs as building blocks of novel stimuli-responsive material systems10. In previous studies, DIBs have proved their ability to respond to electrical9,11, chemical10,12, and optical stimuli13. Various biomolecules with different stimuli-responsive functionalities have been effectively stimulated when reconstituted in the DIB10,14. In light of these successful attempts an important question is raised: could the DIB respond to mechanical stimulus when appropriate biomolecules are incorporated? The interfacial forces acting on a DIB differ from those in other bilayer system15,16. Therefore, the tension in the bilayer held by the droplets could be controlled by regulating tension at the water-lipid-oil interfaces; a concept not applicable with the painted or folded bilayer systems.
MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased membrane tension17,18. In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell19 can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis20. Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique21-23. Reliable structural models explaining MscL&#39;s gating mechanism24,25 are proposed based on its homolog&#39;s crystal structure26,27, modeling28, and results of extensive experimentation 24,29-31. Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming a ~28 &#197; water-filled conductive pore21,24,32. It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel33. Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered22. This property of MscL made possible the design of various controllable valves34, primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs.
In this article, we present an original micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels under mechanical stimulation. We report for the first time, the response of DIBs to mechanical stimulus and the functional reconstitution of the V23T low-threshold mutant of MscL in DIBs35.
The experimental system consists of lipid encased aqueous droplets anchored to the tips of two opposing borosilicate glass micropipettes. When droplets are brought into contact a lipid bilayer interface is formed. This technique offers control over the chemical composition and size of each droplet (bulk), as well as the dimensions of the bilayer interface. In addition, asymmetric membranes with various lipid compositions in each leaflet could be easily formed. Having one of the micropipettes attached to a harmonic piezoelectric actuator, provides the ability to apply a pre-programmed single-cycle or oscillatory stimulus. Tension is delivered to the artificial membrane through the compression of both droplets supporting it. As a result of droplet deformation, the areas of water-lipid-oil interfaces increase, and simultaneously the angle between the droplets decreases, causing an increase in membrane tension and transient MscL activation. Through analysis of the shapes of the droplets during deformation, the tension created at the interface could be estimated. Even though the focus in this article is on the mechano-transduction properties of the DIB, we also emphasize that other types of biomolecules, such as alamethicin, can be activated by this multi-functional platform. We present here, all the technical aspects of preparing, assembling, and taking measurements with this new method in a step-by-step manner.

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Corning</td>
			<td>430624</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-diphytanoy-sn-glycero-3-phosphocholine (DPhPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850356P</td>
			<td>Purchased as lyophilized powder</td>
		</tr>
		<tr>
			<td>34-gauge microfil</td>
			<td>World Precision Instruments</td>
			<td>MF24G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>400 mL Centrifuge bottels</td>
			<td>ThermoFisher</td>
			<td>3141</td>
			<td>Nalgene</td>
		</tr>
		<tr>
			<td>Agilent Function/Arbitrary Waveform Generator, 20 MHz</td>
			<td>Keysight Technologies</td>
			<td>33220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillian</td>
			<td>ThermoFisher</td>
			<td>BP1760</td>
			<td>ACS Grade</td>
		</tr>
		<tr>
			<td>Avanti&#174; Mini-Extruder</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Scope.A1</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>AxioCam HSm</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Pierce&#160;</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>BK Precision 4017B 10 MHz DDs Sweep/Function Generator</td>
			<td>Digi-Key</td>
			<td>BK4017B-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>7 Spectra/Por</td>
			<td>132113</td>
			<td>MWCO 8000, 7.5 mm diameter</td>
		</tr>
		<tr>
			<td>DigiData 1440A system</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>DPhPC</td>
			<td>Avanti</td>
			<td>850356C</td>
			<td></td>
		</tr>
		<tr>
			<td>E-625 PZT Servo-Controller&#160;</td>
			<td>Physik Instrumente</td>
			<td>E-526</td>
			<td></td>
		</tr>
		<tr>
			<td>FPLC System</td>
			<td>Pharmacia Biotech</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>J.T. Baker</td>
			<td>9535-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadecane, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>544-76-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoginizer</td>
			<td>Wheaton</td>
			<td>357426</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Affymetrix</td>
			<td>17886</td>
			<td></td>
		</tr>
		<tr>
			<td>IRGACURE&#174; 2959</td>
			<td>IRGACURE&#174;</td>
			<td>555047962</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopore Membrane Filters&#160;</td>
			<td>EMD Millipore</td>
			<td>VCTP02500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>VWR International</td>
			<td>BDH1133-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td>ACS Grade</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Mallinckrodt</td>
			<td>7100</td>
			<td>ACS Grade</td>
		</tr>
		<tr>
			<td>Kimble-Chase</td>
			<td>Kontes</td>
			<td>420401-1515</td>
			<td>Flex-Column</td>
		</tr>
		<tr>
			<td>LED-100 UV Spot Curing System</td>
			<td>Electro-Lite, corp.</td>
			<td>81170</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Patch-Clamp Micromanipulators</td>
			<td>Thorlabs</td>
			<td>PCS-520N</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>ThermoFisher</td>
			<td>M33</td>
			<td>ACS Grade</td>
		</tr>
		<tr>
			<td>Microelectrode Holder&#160;</td>
			<td>World Precision Instruments</td>
			<td>MEH1S</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS, minimum 99.5% titration</td>
			<td>Sigma-Aldrich</td>
			<td>M1254-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Gas</td>
			<td>Airgas</td>
			<td>UN1066</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>EMD</td>
			<td>SX0420-1</td>
			<td>ACS Grade</td>
		</tr>
		<tr>
			<td>Ni NTA agarose beads</td>
			<td>Qiagen</td>
			<td>1000632</td>
			<td></td>
		</tr>
		<tr>
			<td>Optically Clear Cast Acrylic Tube, 2-1/2&#34; OD x 2&#34; ID</td>
			<td>McMaster-Carr</td>
			<td>8486K545</td>
			<td></td>
		</tr>
		<tr>
			<td>P-601 PiezoMove Flexure-Guided Linear Actuator</td>
			<td>Physik Instrumente</td>
			<td>P-601</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel</td>
			<td>Bio-Rad</td>
			<td>456-9033</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M&#174; All-Purpose Laboratory Film</td>
			<td>Parafilm&#174;</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)1000 dimethacrylate</td>
			<td>Polysciences, Inc.</td>
			<td>15178-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate (PCTE) Membrane Filters, Black, 0.4 Micron, 25mm, 100/Pk</td>
			<td>Sterlitech Corporation</td>
			<td><a href="http://www.sterlitech.com/filters/membrane-disc-filters/polycarbonate-membranes/hydrophilic-polycarbonate-membrane-filters/hydrophilic-polycarbonate-membrane-filter-pctb0425100.html">PCTB0425100</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5405-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder Free Soft Nitrile Examination Gloves&#160;</td>
			<td>VWR International</td>
			<td>CA89-38-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Replacement Gasket 1.0mm</td>
			<td>World Precision Instruments</td>
			<td>GO1-100</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L5750</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>GoodFellow</td>
			<td>147-346-94</td>
			<td>Different diameters could be used depending on the application&#160;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Affymetrix</td>
			<td>21610</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tubes</td>
			<td>ThermoFisher</td>
			<td>14-961-27</td>
			<td>12 x 130 mm</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>ThermoFisher</td>
			<td>BP1421</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracal 30K</td>
			<td>Millipore</td>
			<td>UFC803024</td>
			<td>Amicore Ultra 30 MWCO</td>
		</tr>
		<tr>
			<td>VWR Light-Duty Tissue Wipers</td>
			<td>VWR International</td>
			<td>82003-820</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Scientific 50D Ultrasonic Cleaner</td>
			<td>VWR International</td>
			<td>13089</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Purifier</td>
			<td>Barnstead</td>
			<td>D11931</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>ThermoFisher</td>
			<td>BP1422</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-octylglucopyranoside</td>
			<td>Anatrace</td>
			<td>O311S</td>
			<td></td>
		</tr>
	</tbody>
</table>

multifunctional, micropipette-based method for incorporation and stimulation of bacterial mechanosensitive ion channels in droplet interface bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Figures 1 and 2 display the experimental setup and equipment used to record protein activity in the course of mechanical stimulation of the lipid bilayer membrane. To minimize electrical noise into our measurements, the workstation is placed within a lab-made Faraday cage, grounded to a ground connection on the AxoPatch 200 B Amplifier.
Formation of a stable insulating lipid bilayer is a key step in this study. In this arrangement, a lipid monolayer assembles at the oil/water interface of the aqueous droplets immersed in a bath of an organic solvent. When droplets are placed in contact, excess oil is eliminated, and the opposing lipid monolayers thin to a two-molecule thick lipid bilayer. The most common technique used in bilayer characterization is voltage-clamp. With voltage-clamp, the voltage across the bilayer is maintained at a constant value while the current is measured. Figure 4 portrays a typical real-time current recording of the initial bilayer formation. Knowing the specific capacitance (~0.6 &#956;F/cm2)5 of the DPhPC lipid bilayer, the area of the formed bilayer could be calculated. The bilayer area could be controlled by changing the position of the droplets (Figure 4A). Using the piezoelectric actuator, different types of waveforms (sinusoidal, square, triangular, etc.) at different frequencies, amplitudes, and duty cycles could be applied to the droplets to horizontally and axially oscillate them and thus, bilayer tension and area could be altered (Figure 4B).
When the DIB is mechanically stimulated, while maintaining a constant DC potential across the membrane, a low-threshold (gain-of-function) V23T mutant of MscL generates reliable activities including mainly sub-conductive states and occasionally full opening events (Figure 5). These events are identical to those recorded using the patch-clamp technique from intact inner E. coli membranes and liposomes reconstituted with the purified V23T MscL. The results in Figure 5 prove that gating occurs in response to an increase in tension, since all current spikes are observed at peak compression. At peak compression, the relative areal expansion of the droplets is maximal and therefore, tension at the interface is maximal.
Alamethicin, a voltage-gated ion channel and one of the most studied peptides, increases the membrane permeability when a DC voltage is applied across the membrane36. The ability of the lipid bilayer interface to host transmembrane proteins and peptides is also tested by performing voltage-gating current recordings using alamethicin peptide. Alamethicin is mixed with the phospholipid solution to a final concentration of 100 ng/ml. Figure 6 shows the current measurements under voltage clamp (+115 mV). The droplets in this experiment are pulled apart in order to achieve small bilayer interface and thus higher resistance and smaller capacitance. The gating behavior of the Alamethicin peptide is shown through the discrete steps of current (Figure 6). The histogram on the right side of the plot shows the changes in conductance from the base level (0.0962 nS), which is basically the first conductance level of the channel itself.
<img alt="Figure 1" src="/files/ftp_upload/53362/53362fig1.jpg" /> 
Figure 1: A schematic describing the main parts and dimensions of the oil reservoir. The oil reservoir is manufactured at the machine shop at Virginia Tech. It consists of a machined cylindrical acrylic tube glued to the surface of an acrylic sheet. The dimensions and design can be modified to accommodate different applications or more than two micropipettes. <a href="https://www.jove.com/files/ftp_upload/53362/53362fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53362/53362fig2.jpg" /> 
Figure 2: Experimental setup and micropipettes preparation. (A) The standard workstation for forming, mechanically stimulating, and characterizing the interface bilayers includes a microscope, 3-axis manipulators, a digital camera, piezoelectric oscillator, vibration isolation table, and a Faraday cage (not shown). (B) The experimental setup consists of two opposing PEG-DMA hydrogel filled micropipettes horizontally positioned within a bath of Hexadecane oil. Each of the micropipettes contains an Ag/AgCl electrode to provide electrical connection. A third micropipette filled with proteoliposome solution is used to form the droplets at the tip of the other micropipettes. (C) The DIB current response could be measured using a combination of the patch amplifier and the low-noise data acquisition system. (D) A closed up picture showing the aqueous droplets formed at the tip of the micropipettes. (E) Ag/AgCl electrodes are made by dipping the tip of two 250 &#956;m silver wires in bleach. The electrodes are then fed through two borosilicate glass capillaries filled with PEG-DMA hydrogel, which is cured with UV light to solidify. A straight microelectrode holder with male connector is used to connect one of the micropipettes to the headstage of the patch amplifier.
<img alt="Figure 3" src="/files/ftp_upload/53362/53362fig3.jpg" /> 
Figure 3: Images illustrating the formation of droplet interface bilayers. (A) A 10 &#956;m micropipette filled with proteoliposomes is positioned under the microscope in proximity to the micropipette tips. Using a syringe connected to the micropipette, dispense small volumes of the proteoliposomes to form spherical droplets to desired volume. Let the monolayer form by allowing the droplets to sit for ten min. Bring the droplets into contact; the bilayer will form after all oil at the interface is eliminated. (B) While the bilayer is formed, the chemical composition at both sides of the interface could be controlled by injecting desired chemicals using a micro-sized micropipette. (C) The droplets at the moment of first contact. (D) The droplets when the lipid bilayer is formed.
<img alt="Figure 4" src="/files/ftp_upload/53362/53362fig4.jpg" /> 
Figure 4: Real-time measurements show both the initial thinning and subsequent expansion of the interface. (A) Current measured in the course of bilayer formation through the application of a triangular electrical potential. The magnitude of the measured current is directly proportional to the capacitance, and thus the area of the bilayer interface. The closer the droplets are brought together, the bigger the area of the interface and vice versa. (B) Upon application of mechanical excitation, the area of the bilayer interface increases and decreases at the same frequency as the stimulating signal.
<img alt="Figure 5" src="/files/ftp_upload/53362/53362fig5.jpg" /> 
Figure 5: Real-time measurements show the response of the bilayer to mechanical excitation as well as the gating of the V23T mutant of MscL. The shape of the current response is sinusoidal, which relates to a sinusoidal change in bilayer capacitance as a result of the bilayer area change. The current spikes, occurring at the peak of each cycle, indicate sub-conductance gating of the V23T mutant. A polar plot further indicates that gating occurs at peak compression, which reflects an increase in tension at the bilayer interface.
<img alt="Figure 6" src="/files/ftp_upload/53362/53362fig6.jpg" /> 
Figure 6: Current measurements under voltage clamp and corresponding histogram of conductance levels for gating activity of incorporated Alamethicin channels. The gating behavior of the Alamethicin peptide is shown through the discrete step-wise increase in current. The conductance levels match very well with previous measurements performed by our research group at Virginia Tech7.

Watch this Scientific Journal Video about Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers at JoVE.

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

multifunctional-micropipette-based-method-for-incorporation

Research

JoVE Journal

Biology

10.7K 조회수.  Virginia Polytechnic Institute and State University.  세균 mechanosensitive 채널은 생체 분자 장치에서 mechanoelectrical 트랜스 듀서로 사용할 수 있습니다. 물방울 인터페이스 이중층 (DIB의), 이러한 장치에 휴대 영감 빌딩 블록, 통합 및 mechanosensitive 채널을 자극하는 새로운 플랫폼을 나타냅니다. 여기서 우리는, DIB의 형성 기계적인 자극에서 mechanosensitive 채널의 연구를 허용하는 새로운 마이크로 피펫 기반 방법을 보?...

비디오: Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers