Name: Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles (Video) | JoVE Journal
Uploaded: 2024-04-12T14:10:00.000+00:00
Duration: 10 min 56 s
Description: Discover methods for constructing synthetic cells using membrane protein reconstitution and enzyme encapsulation in lipid vesicles. This protocol enables autonomous growth systems for applications in diagnostics and therapy.

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The authors thank Aditya Iyer for the cloning of the pBAD-PercevalHR gene and Gea Schuurman-Wolters for aiding with protein production and purification. The research was funded by the NWO Gravitation program &#34;Building a Synthetic Cell&#34; (BaSyC).

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

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</ol>

The authors declare no competing financial interest.

We present a protocol for the synthesis of (membrane) protein containing sub-micrometer size lipid vesicles (proteoLUVs), and the conversion of proteoLUVs into giant-unilamellar vesicles (proteoGUVs). The protocol should be applicable for the reconstitution of other membrane proteins13,19,30,40 and the encapsulation of metabolic networks other than the L-arginine breakdown and glycerol 3-phosphate synthesis pathways presented here.
The reconstitution of membrane proteins in liposomes generally yields a random orientation of the proteins within the lipid membrane. For ArcD and Glpt, the orientation does not matter, because the proteins operate bidirectionally and the solutes move depending on the sign of the total electrochemical potential. For proteins that function in one direction, half of the molecules will not be available for activity when their orientation is 50/50.
The extrusion of vesicles through a polycarbonate filter narrows the size distribution, and the vesicles become more homogeneous the smaller the pore size of the filters. However, smaller vesicles come at the price of having a smaller volume and thus, a very small number of proteins (enzymes, membrane proteins) per vesicle. To minimize the fraction of LUVs having one or more components missing, the concentration of the proteins can be adjusted as shown in Figure 3A,B but with very small vesicles stochastic effects are unavoidable.
The gel-assisted swelling method relies on the deposition of sonicated vesicles (SUVs) on a dried agarose surface. During the drying process, a concentration gradient of vesicles is formed and the lipid concentration is the highest at the periphery of the spot. This is due to a phenomenon known as the coffee stain&#160;effect41,42. As a result, only a small region within the spot contains a concentration of lipids suitable for GUV formation. Circular uneven-concentrated lipid spots result in large unused areas that are not available for vesicle fusion; hence, the efficiency of GUV formation becomes low in this approach. To increase the yield of GUVs, one should aim for a uniform lipid deposition, which can be accomplished by spin coating42 or leveraging the coffee stain effect41.
Further notes on the protocol:
Care should be taken that several membrane proteins (&#62;10) are reconstituted on average per vesicle to avoid stochastic effects. Thus, avoid lipid-to-protein ratios higher than 400:1 w/w. A random orientation is assumed for most proteins.
Ideally, the volume of total membrane proteins is as small as possible and does anyway not exceed 10-20% of the liposome volume. Adding larger volumes will introduce more detergent and the majority of preformed liposomes may lyse, which can have a negative effect on the reconstitution efficiency.
Pre-equilibration of the extruder with the appropriate solution is important to avoid dilution of the components during the encapsulation. Ideally, the preequilibration solution should also contain the soluble enzymes, but including enzymes can be too costly due to the relatively large volume needed for the pre-equilibration of the extruder. Therefore, we typically omit these components and accept a 5% dilution of the enzymes.
Ionophores should be added to the mixtures containing proteoliposomes because the molecules are highly hydrophobic and may adsorb to surfaces if vesicles are not present. Care should be taken that the volume of solvent added is low (&#60;1% of total reaction volume). Controls are done to verify that the solvents do not affect the metabolic networks in the vesicles. Ionophore concentrations of about 1 &#181;M are generally safe for total lipid concentrations of ~3 mg/mL.
Care should be taken to dry the droplets thoroughly. If the droplets are not dry enough, GUV formation is less efficient as lipids will form lipid aggregates and MLVs may form when the swelling solution is added.
Glucose is used to match the osmolarity of the external to the internal medium; the latter contains sucrose instead of glucose to increase the density of the vesicles and thereby facilitate the sedimentation of the GUVs. Moreover, glucose in the external environment enhances the phase contrast, making the vesicle more easily visible.
Stochastic effects are much less of a problem with GUVs; but here, the surface-to-volume ratio can become unacceptably low such that the number of membrane proteins (e.g., transporters) becomes limiting for the internal metabolic network. The method described in this protocol does not allow precise control over the size of the proteoGUVs but the majority fall in the size range of 5-50 &#181;m.
In conclusion, the work presented here provides a comprehensive overview of membrane protein reconstitution and enzyme encapsulation in lipid vesicles of varying sizes. We demonstrate the construction of functional vesicles for the synthesis of ATP and building blocks from simple precursors such as amino acids and glycerol. Reconstituting membrane proteins in GUVs remains challenging, but the here-presented protocols pave the way for further developments in bottom-up synthetic biology research.

The field of bottom-up synthetic biology focuses on constructing (minimal) cells1,2 and metabolic bioreactors for biotechnological3,4 or biomedical purposes5,6,7,8. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical functions of proteins and reaction networks9. As an intermediate step towards an autonomously functioning synthetic cell, modules are developed that capture essential features of living cells such as metabolic energy conservation, protein and lipid synthesis, and homeostasis. Such modules not only enhance our understanding of life but also have potential applications in the fields of medicine8 and biotechnology10.
Transmembrane proteins are at the heart of virtually any metabolic network as they transport molecules in or out of the cell, signal, and respond to the quality of the environment, and play numerous biosynthetic roles. Thus, the engineering of metabolic modules in synthetic cells requires in most cases the reconstitution of integral and/or peripheral membrane proteins into a membrane bilayer composed of specific lipids and high integrity (low permeability). The handling of these membrane proteins is challenging and requires specific knowledge and experimental skills.
Several methods have been developed to reconstitute membrane proteins within phospholipid vesicles, most often with the purpose of studying the function11,12, regulation13, kinetic properties14,15, lipid dependence15,16, and/or stability17 of a specific protein. These methods involve the rapid dilution of detergent-solubilized protein into aqueous media in the presence of lipids18, the removal of detergents by incubating detergent-solubilized protein with detergent-destabilized lipid vesicles and absorption of the detergent(s) onto polystyrene beads19, or the removal of detergents by dialysis or size-exclusion chromatography20. Organic solvents have been used to form lipid vesicles, for example, via the formation of oil-water interphases21, but the majority of integral membrane proteins are inactivated when exposed to such solvents.
In our laboratory, we mostly reconstitute membrane proteins by the detergent-absorption method to form large-unilamellar vesicles (LUVs)19. This method allows the co-reconstitution of multiple membrane proteins and the encapsulation in the vesicle lumen of enzymes, metabolites, and probes22,23. The membrane protein-containing LUVs can be converted into giant-unilamellar vesicles (GUVs) with/without encapsulation of water-soluble components, using either electroformation24 or gel-assisted swelling25 and specific conditions to preserve the integrity of the membrane proteins26.
This paper presents a protocol for the reconstitution in LUVs of an out-of-equilibrium metabolic network that regenerates ATP through the breakdown of L-arginine into L-ornithine27. The formation of ATP is coupled to the production of glycerol-3-phosphate (G3P), an important building block for phospholipid synthesis22,28. The metabolic pathway consists of two integral membrane proteins, an arginine/ornithine (ArcD) and a G3P/Pi antiporter (GlpT). In addition, three soluble enzymes (ArcA, ArcB, ArcC) are required for the recycling of ATP, and GlpK is used to convert glycerol into glycerol 3-phosphate, using the ATP from the breakdown of L-arginine, see Figure 1 for a schematic overview of the pathway. This protocol represents a good starting point for the future construction of even more complex reaction networks-for the synthesis of lipids or proteins or the division of cells. The lipid composition of the vesicles supports the activity of a wide variety of integral membrane proteins and has been optimized for the transport of diverse molecules into or out of the vesicles27,29,30.
<img alt="Metabolic pathway diagram of L-arginine to L-citrulline, involving ArcA, ArcB enzymes in cells." class="xfigimg" src="/files/ftp_upload/66627/66627fig01v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In short, purified membrane proteins (solubilized in dodecyl-β-D-maltoside, DDM) are added to preformed lipid vesicles that have been destabilized with Triton X-100, which allows the insertion of the proteins into the membrane. The detergent molecules are subsequently (slowly) removed by the addition of activated polystyrene beads, resulting in the formation of well-sealed proteoliposomes. Soluble components can then be added to the vesicles and encapsulated via freeze-thaw cycles, which traps the molecules in the process of membrane fusion. The obtained vesicles are highly heterogeneous and many are multilamellar. They are then extruded through a polycarbonate filter with a pore size of 400, 200, or 100 nm, which yields more uniformly sized vesicles; the smaller the pore size, the more homogeneous and unilamellar the vesicles but at the price of a smaller internal volume. Non-incorporated proteins and small molecules are removed from the external solution by size-exclusion chromatography. The proteoLUVs can be converted into micrometer size vesicles by gel-assisted swelling, and these proteoGUVs are then collected and trapped in a microfluidic chip for microscopic characterization and manipulation. Figure 2 shows a schematic overview of the full protocol.
<img alt="Proteoliposome formation diagram; detergent titration process; microscopy images for activity assays." class="xfigimg" src="/files/ftp_upload/66627/66627fig02v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Overview of the protocol for reconstituting membrane proteins and encapsulating enzymes and water-soluble components in lipid vesicles of sub-micrometer (LUVs) and micrometer size (GUVs). <a href="https://www.jove.com/files/ftp_upload/66627/66627fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The reconstitution and encapsulation protocols work well and the functionality of the proteins is retained, but the proteoLUVs and proteoGUVs are heterogeneous in size. Microfluidic approaches31,32 allow the formation of micrometer-sized vesicles that are more homogeneous in size, but functional reconstitution of membrane proteins is generally not possible because residual solvent in the bilayer inactivates the proteins. The proteoLUVs range in size from 100 to 400 nm, and at low concentrations of enzymes, the encapsulation may lead to vesicles with incomplete metabolic pathways (stochastic effects; see Figure 3). LUVs are ideal for constructing specific metabolic modules, as shown here for the production of ATP and building blocks like G3P. Such proteoLUVs can potentially be encapsulated in GUVs and serve as organelle-like compartments for the host vesicles.
<img alt="Protein distribution graphs A: soluble proteins vs μM; B: membrane proteins vs LPR; size comparison." class="xfigimg" src="/files/ftp_upload/66627/66627fig03v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Number of molecules per vesicle with a diameter of 100, 200, or 400 nm. (A) When the encapsulated proteins (enzymes, probes) are in the range of 1-10 µM. (B) The reconstitution is done at 1 to 1,000, 1 to 10,000, and 1 to 100,000 membrane proteins per lipid (mol/mol). We make the assumption that molecules are encapsulated at the indicated concentrations and incorporated in the membrane at these protein-to-lipid ratios. For some enzymes, we have seen that they bind to membranes, which can increase their apparent concentration in the vesicles. Abbreviation: LPR = Lipid-Protein-Ratio <a href="https://www.jove.com/files/ftp_upload/66627/66627fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9414-25g</td><td></td></tr><tr><td>Amicon cut-off filter</td><td>Sigma Aldrich</td><td>Milipore centrifugal filter units Amicon Ultra&amp;nbsp;</td><td></td></tr><tr><td>BioBeads</td><td>BioRad</td><td>152-3920</td><td></td></tr><tr><td>CHCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Macron Fine Chemicals</td><td>MFCD00000826</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Formedium</td><td>-</td><td></td></tr><tr><td>D(+)-Sucrose</td><td>Formedium</td><td>-</td><td></td></tr><tr><td>DDM</td><td>Glycon</td><td>D97002 -C</td><td></td></tr><tr><td>Diethyl Ether</td><td>Biosolve</td><td>52805</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855-100ml</td><td></td></tr><tr><td>DOPC</td><td>Avanti</td><td>850375P-1g</td><td></td></tr><tr><td>DOPE</td><td>Avanti</td><td>850725P-1g</td><td></td></tr><tr><td>DOPG</td><td>Avanti</td><td>840475P-1g</td><td></td></tr><tr><td>DTT</td><td>Formedium&amp;nbsp;</td><td>DTT005</td><td></td></tr><tr><td>EtOH</td><td>J.T.Baker Avantor</td><td>MFCD00003568</td><td></td></tr><tr><td>Extruder</td><td>Avestin Inc</td><td>LF-1</td><td></td></tr><tr><td>Fluorimeter</td><td>Jasco</td><td>Spectrofluorometer FP-8300</td><td></td></tr><tr><td>Glycerol</td><td>BOOM</td><td>51171608</td><td></td></tr><tr><td>Gravity flow column</td><td>Bio-Rad</td><td>732-1010</td><td></td></tr><tr><td>Hamilton syringe 100 &amp;micro;L</td><td>Hamilton</td><td>7656-01</td><td></td></tr><tr><td>Hamilton syringe 1000 &amp;micro;L</td><td>Hamilton</td><td>81320</td><td></td></tr><tr><td>Handheld LCP dispenser</td><td>Art Robbins Instruments</td><td>620-411-00</td><td></td></tr><tr><td>Handheld Sonicator</td><td>Hielscher Ultrasound Technology</td><td>UP50H</td><td></td></tr><tr><td>HCl</td><td>BOOM</td><td>x76021889.1000</td><td></td></tr><tr><td>Imidazole</td><td>Roth</td><td>X998.4-250g</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Supelco</td><td>1.05099.1000</td><td></td></tr><tr><td>KCl</td><td>BOOM</td><td>76028270.1</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Supelco</td><td>1.04873.1000</td><td></td></tr><tr><td>Kimwipe</td><td>Kimtech Science</td><td>7552</td><td></td></tr><tr><td>Large Falcon tube centrifuge</td><td>Eppendorf</td><td>Centrifuge 5810 R</td><td></td></tr><tr><td>L-Arginine</td><td>Sigma-Aldrich</td><td>A5006-100G</td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td>DM LS2</td><td></td></tr><tr><td>L-Ornithine</td><td>Roth</td><td>T204.1</td><td></td></tr><tr><td>LSM Laser Scanning Confocal Microscope</td><td>Zeiss</td><td>LSM 710 ConfoCor 3</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2670-1KG</td><td></td></tr><tr><td>Microfluidic chip</td><td>Homemade&amp;nbsp;</td><td>PDMS based</td><td>DOI: https://doi.org/10.1039/C8LC01275J</td></tr><tr><td>Na-ADP</td><td>Sigma-Aldrich</td><td>A2754-1G</td><td></td></tr><tr><td>NaCl</td><td>Supelco</td><td>1.06404.1000</td><td></td></tr><tr><td>Nanodrop Spectrometer</td><td>Isogen Life Science</td><td>ND-1000 spectrophotometer NanoDrop</td><td></td></tr><tr><td>NaOH</td><td>Supelco</td><td>1.06498.1000</td><td></td></tr><tr><td>Needles for GUVs</td><td>Henke-Ject</td><td>14-14575</td><td>27 G x 3/4&#039;&#039; 0.4 x 20 mm</td></tr><tr><td>Needles for microfluidics</td><td>Henke-Ject</td><td>14-15538</td><td>18 G x 1 1/2&#039;&#039; 1.2 x 40 mm</td></tr><tr><td>Ni&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; Sepharose</td><td>Cytiva</td><td>17526802</td><td></td></tr><tr><td>Nigericin</td><td>Sigma-Aldrich</td><td>N7143-5MG</td><td></td></tr><tr><td>Nutator</td><td>VWR</td><td>83007-210</td><td></td></tr><tr><td>Osmolality meter</td><td>Gonotec Salmenkipp</td><td>Osmomat 3000 basic freezing point osmometer</td><td></td></tr><tr><td>Plasmacleaner</td><td>Plasma Etch</td><td>PE-Avenger</td><td></td></tr><tr><td>Polycarbonate filter</td><td>Cytiva Whatman</td><td>Nuclepor Track-Etch Membrane Product: 10417104</td><td>0.4 &amp;micro;m</td></tr><tr><td>Polycarbonate ultracentrifuge tube</td><td>Beckman Coulter</td><td>355647</td><td></td></tr><tr><td>Pyranine</td><td>Acros Organics</td><td>H1529-1G</td><td></td></tr><tr><td>Quartz cuvette (black)</td><td>Hellma Analytics</td><td>108B-10-40</td><td></td></tr><tr><td>Sephadex G-75 resin&amp;nbsp;</td><td>GE Healthcare</td><td>17-0050-01</td><td></td></tr><tr><td>Sonicator</td><td>Sonics Sonics &amp;amp; Materials INC</td><td>Sonics vibra cell</td><td></td></tr><tr><td>Syringe filter</td><td>Sarstedt</td><td>Filtropur S plus 0.2</td><td>0.2 &amp;micro;m</td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>A-42467</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Eppendorf</td><td>centrifuge 5418</td><td></td></tr><tr><td>Teflon spacer</td><td>Homemade&amp;nbsp;</td><td>Teflon based</td><td>45 x 26 x 1.5 or 45 x 26 x 3 or 20 x 20 x 3 mm</td></tr><tr><td>Tris</td><td>PanReac AppliChem</td><td>A1086.1000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787-100 ml</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima Max-E</td><td></td></tr><tr><td>UV lamp</td><td>Spectroline</td><td>ENB-280C/FE</td><td></td></tr><tr><td>UV/VIS Spectrometer</td><td>Jasco</td><td>V730 spectrophotometer</td><td></td></tr><tr><td>Valinomycin</td><td>Sigma-Aldrich</td><td>V0627-10MG</td><td></td></tr><tr><td>Widefield fluorescence microscope</td><td>Zeiss</td><td>AxioObserver</td><td></td></tr><tr><td>&amp;beta;-Casein</td><td>Sigma Aldrich</td><td>C5890-500g</td><td></td></tr></tbody></table>

construction of out&#45;of&#45;equilibrium metabolic networks in nano&#45; and micrometer&#45;sized vesicles

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

The reconstitution of solubilized membrane proteins in liposomes requires the destabilization of preformed vesicles. The addition of low amounts of Triton X-100 initially results in an increase of absorbance at 540 nm (A540) due to an increase in light scattering by the swelling of the vesicles (Figure 4). The maximum A540 value is the point where the liposomes are saturated with detergent (Rsat), after which any further addition of Triton X-100 will result in partial solubilization of the vesicles. At Rsol, the liposomes are completely solubilized (Figure 4). For the reconstitution of membrane proteins, we typically use vesicles destabilized to 60% beyond Rsat (indicated by arrow).
The reconstituted membrane protein ArcD is used to transport L-arginine into and L-ornithine out of the vesicles and is used together with the encapsulated enzymes ArcA, ArcB, and ArcC to recycle ATP from ADP and inorganic phosphate. GlpT is a glycerol 3-phosphate/phosphate antiporter that together with GlpK is needed for the synthesis (and excretion) of glycerol 3-phosphate from glycerol plus ATP produced by the L-arginine breakdown. The addition of 5 mM L-arginine at t = 0 to the proteoliposomes results in a steep increase in the ratio of the PercevalHR fluorescence at 500 and 430 nm excitation39, which reflects the ATP/ADP ratio inside the vesicles (Figure 5). Upon addition of glycerol, the ATP is used for the synthesis of glycerol 3-phosphate, which results in a lowering of the ATP/ADP ratio28. The breakdown of L-arginine also leads to pH changes, which can be quantified using pyranine or pHluorin27.
<img alt="L-arginine fluorescence ratio graph; F500/F430 over time; optical emission analysis results." class="xfigimg" src="/files/ftp_upload/66627/66627fig05v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Recycling of ATP by the L-arginine breakdown pathway. LUVs at 2.7 mg of lipid/mL are placed in a cuvette and the ratio of fluorescence at 500 nm and 430 nm is recorded. 5 mM of L-arginine is added at t = 0. The F500/F430 ratio is a measure of the ATP/ADP ratio inside the vesicles. Abbreviation: LUVs = large-unilamellar vesicles. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The ATP-producing LUVs can also be used to form GUVs. Rehydration of dried proteo-LUVs on an LGT agarose gel results in the formation of micrometer-size vesicles (Figure 6). The formation of proteo-GUVs within the dried LUV spots heavily depends on the local concentration of lipids and the degree of dehydration. Some areas have large patches of GUVs, whereas at other places in the same droplet, no or few GUVs can be formed. The formation of GUVs via LGT agarose-assisted swelling results in a range of GUV sizes, typically from a few micrometers to more than 50 μm.
<img alt="Microscope image, liquid crystals textures, optical study, phase transition observation, scale bars shown." class="xfigimg" src="/files/ftp_upload/66627/66627fig06v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: DIC images of proteoGUVs formed by gel-assisted swelling. Scale bar = 25 µm. Abbreviations: DIC = differential interference contrast; GUVs = giant-unilammellar vesicles. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The GUVs are harvested and trapped in a β-casein passivated PDMS-based microfluidic device (Figure 7). The device is designed in a way that many vesicles can be trapped and analyzed within a single experiment, and the device is used to control the external environment (flow rate, buffer composition, etc.)34. Even when the yield of proteo-GUVs is low, the constant flow of GUVs through the microfluidic chip enables many vesicles to be collected. Once enough GUVs are trapped, the system is washed with an osmotically balanced buffer to remove external components such as soluble proteins, small molecules, and fluorescent probes. The washing buffer is then replaced by a substrate solution of equal osmolality containing 50 mM KPi (pH 7.0), varying amounts of glucose (to adjust the osmolality), and substrates such as 10 mM L-arginine. A time series experiment is done on several traps of the microfluidic chip and images are taken every 90 s (405 nm and 488 nm excitation when PercevalHR is used). Further, a brightfield image is taken at each time point. The ratio of the emission intensities after excitation at 488 and 405 nm is a measure of the ADP/ATP ratio in the vesicles.
<img alt="Static equilibrium diagram; microscopy images at 405 nm, bright field, 488 nm; optical flow analysis." class="xfigimg" src="/files/ftp_upload/66627/66627fig07.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
Figure 7: Trapping of vesicles with the microfluidic chip. (A) Trapping of the vesicles, accompanied by the ability to control the external environment and flow rate. (B) ProteoGUVs trapped in a microfluidic device and excited with a 405 nm laser (green) and 488 laser (red). Emission for both channels is measured at &gt;500 nm. The brightfield image allows for visualization of the vesicles without fluorescence. Scale bar = 25 µm. Abbreviation: GUVs = giant-unilamellar vesicles. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

construction-out-equilibrium-metabolic-networks-nano-micrometer-sized

Research

JoVE Journal

Biochemistry

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

This article introduces a simple one-pot reconstitution of cytoskeletal networks inside phase separated giant vesicles. The method can be generalized and applied to encapsulation or confinement of a wide range of biomolecules.

In vitro Reconstitution of Cytoskeletal Networks inside Phase Separated Giant Unilamellar Vesicles (GUVs)

Discover methods for constructing synthetic cells using membrane protein reconstitution and enzyme encapsulation in lipid vesicles. This protocol enables autonomous growth systems for applications in diagnostics and therapy.

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

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