Layer-by-layer Cell Membrane AssemblySandro Matosevic and Brian M. Paegel*Brian M. PaegelPh.D., Chemistry, University of California, Berkeley, 2003B.S., Chemistry, Duke University, 1998Assistant Professor, Scripps Research Institute

IntroductionPhospholipid membranes are complex supramolecular assemblies involved in every aspect of biological function.Important traits to replicate include curvature, fluidity, lamellarity, and composition.Current methods:Bulk assembly without control over vesicle size, content, membrane composition, and lamellarity.Microfluidic vesicle assembly allows control over size variation and can encapsulate cargo with 80% efficiency.Deposition based strategy that controls single layer vesicle starting with lipid-stabilized water-in-oil emulsions.MethodDroplets of oil are emulsified into water droplets and then centrifuged.This makes them cross a oil/water phase boundary.Lipid droplets are added to each water-in-oil droplet and stick to it.This adds a layer of lipids to each droplet every time the water droplet crosses a phase boundary with the oil. This adds several layers of lipids and needed content using a microfluidic droplet array.

Method Cont.The droplet array uses 3 fluidic input lines.Oil-lipid mixture of dioleoylphosphatidylcholine (DOPC) dissolved in squalineCytoplasmic aqueous phase of TAE buffer to fill the vesicles - AQcyExtracellular aqueous phase - AQex

ResultsTest unilamellar presence after single deposition:Test perfuses haemolysin into cells to act as channels. Different sized flourescent dyes are added and only small dye goes into molecule.Same permeability with vesicles loaded with DNA that encodes for haemolysin gene.Haemolysin only permeates single bilayers. Proves unilamellar structure.

Results Cont.Test the quantities of material going into each monolayer formed during the synthesis of the membrane.Add fluorescent reporter lipids into each monolayer addition to test quantities.If added correctly, fluorescence should increase linearly.Final cooling and discard of waste should result in a fraction of final fluorescence. Inversely proportional to number of depositions.

ConclusionLipid bilayer assembly via deposition is now a viable route to asymmetric unilamellar and double-bilayer membranes.Unilamellar vesicles can support functional insertion and pore-forming protein complexes, and support a basic cell-like metabolism.ReferencesSandro Matosevic &Brian M. Paegel. (2013). Layer-by-layer cell membrane assembly. Nature Chemistry5,958963.

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