Abstract
Droplet interface bilayers (DIBs) are an emerging tool within synthetic biology that aims to recreate biological processes in artificial cells. A critical component for the utility of these bilayers is controlled flow between compartments and, notably, uphill transport against a substrate concentration gradient. A versatile method to achieve the desired flow is to exploit the specificity of membrane proteins that regulate the movement of ions and transport of specific metabolic compounds. Methods have been in existence for some time to synthesize proteins within a droplet as well as incorporate membrane proteins into DIBS; however, there have been few reports combining synthesis and DIB incorporation for membrane transporters that demonstrate specific, uphill transport. This chapter presents two methods for the incorporation of a membrane transporter into a simple two-droplet DIB system, with the downhill and uphill transport reaction readily monitored by fluorescence microscopy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kurihara K, Tamura M, Shohda K et al (2011) Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nat Chem 3(10):775–781. https://doi.org/10.1038/nchem.1127
Peters RJRW, Marguet M, Marais S et al (2014) Cascade reactions in multicompartmentalized polymersomes. Angew Chem Int Ed Engl 53(1):146–150. https://doi.org/10.1002/anie.201308141
Bayley H, Cronin B, Heron A et al (2008) Droplet interface bilayers. Mol BioSyst 4(12):1191–1208. https://doi.org/10.1039/b808893d
Leptihn S, Castell OK, Cronin B et al (2013) Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nat Protoc 8(6):1048–1057. https://doi.org/10.1038/nprot.2013.061
Findlay HE, Harris NJ, Booth PJ (2016) In vitro synthesis of a major facilitator transporter for specific active transport across droplet interface bilayers. Sci Rep 6:39349. https://doi.org/10.1038/srep39349
Carreras P, Law RV, Brooks N et al (2014) Microfluidic generation of droplet interface bilayer networks incorporating real-time size sorting in linear and non-linear configurations. Biomicrofluidics 8(5):054113. https://doi.org/10.1063/1.4897495
Villar G, Graham AD, Bayley H (2013) A tissue-like printed material. Science 340(6128):48–52. https://doi.org/10.1126/science.1229495
Haylock S, Friddin MS, Hindley JW et al (2020) Membrane protein mediated bilayer communication in networks of droplet interface bilayers. Commun Chem 3(1):77. https://doi.org/10.1038/s42004-020-0322-1
Holden MA, Needham D, Bayley H (2007) Functional bionetworks from nanoliter water droplets. J Am Chem Soc 129(27):8650–8655. https://doi.org/10.1021/ja072292a
Elfaramawy MA, Fujii S, Uyeda A et al (2018) Quantitative analysis of cell-free synthesized membrane proteins at the stabilized droplet interface bilayer. Chem Commun 54(86):12226–12229. https://doi.org/10.1039/c8cc06804f
Venkatesan GA, Lee J, Farimani AB et al (2015) Adsorption kinetics dictate mono layer self-assembly for both lipid-in and lipid-out approaches to droplet Interface bilayer formation. Langmuir 31(47):12883–12893. https://doi.org/10.1021/acs.langmuir.5b02293
Hwang WL, Chen M, Cronin B et al (2008) Asymmetric droplet interface bilayers. J Am Chem Soc 130(18):5878–5879. https://doi.org/10.1021/ja802089s
Syeda R, Holden MA, Hwang WL et al (2008) Screening blockers against a potassium channel with a droplet interface bilayer array. J Am Chem Soc 130(46):15543–15548. https://doi.org/10.1021/ja804968g
Booth MJ, Schild VR, Graham AD et al (2016) Light-activated communication in synthetic tissues. Sci Adv 2(4):e1600056. https://doi.org/10.1126/sciadv.1600056
Kaback HR, Smirnova I, Kasho V et al (2011) The alternating access transport mechanism in LacY. J Membr Biol 239(1–2):85–93. https://doi.org/10.1007/s00232-010-9327-5
Miller D, Booth PJ, Seddon JM et al (2013) Protocell design through modular compartmentalization. J R Soc Interface 10(87):20130496. https://doi.org/10.1098/rsif.2013.0496
Dowhan W, Bogdanov M (2011) Lipid-protein interactions as determinants of membrane protein structure and function. Biochem Soc Trans 39(3):767–774. https://doi.org/10.1042/BST0390767
Vitrac H, Bogdanov M, Dowhan W (2013) In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein. Proc Natl Acad Sci 110(23):9338–9343. https://doi.org/10.1073/pnas.1304375110
Allen-Benton M, Findlay HE, Booth PJ (2019) Probing membrane protein properties using droplet interface bilayers. Exp Biol Med (Maywood) 244(8):709–720. https://doi.org/10.1177/1535370219847939
Findlay HE, Booth PJ (2017) The folding, stability and function of lactose permease differ in their dependence on bilayer lipid composition. Sci Rep 7(1):13056. https://doi.org/10.1038/s41598-017-13290-7
Sandermann H Jr (1977) beta-D-Galactoside transport in Escherichia coli: substrate recognition. Eur J Biochem 80(2):507–515. https://doi.org/10.1111/j.1432-1033.1977.tb11906.x
Harris NJ, Findlay HE, Simms J et al (2014) Relative domain folding and stability of a membrane transport protein. J Mol Biol 426(8):1812–1825. https://doi.org/10.1016/j.jmb.2014.01.012
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675. https://doi.org/10.1038/nmeth.2089
Dubois M, Gilles KA, Hamilton JK et al (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–356. https://doi.org/10.1021/ac60111a017
Markwell MAK, Haas SM, Bieber LL et al (1978) Modification of Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87(1):206–210. https://doi.org/10.1016/0003-2697(78)90586-9
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Findlay, H.E., Harris, N.J., Booth, P.J. (2021). Integrating Membrane Transporter Proteins into Droplet Interface Bilayers. In: Moreira, I.S., Machuqueiro, M., Mourão, J. (eds) Computational Design of Membrane Proteins. Methods in Molecular Biology, vol 2315. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1468-6_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1468-6_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1467-9
Online ISBN: 978-1-0716-1468-6
eBook Packages: Springer Protocols