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

<ol>
	<li>Hirschi, S., Ward, T. R., Meier, W. P., M&#252;ller, D. J., Fotiadis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology:+bottom-up+assembly+of+molecular+systems.">Synthetic biology: bottom-up assembly of molecular systems.</a> Chem Rev. 122 (21), 16294-16328 (2022).</li><li>Ivanov, I., 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=Bottom-up+synthesis+of+artificial+cells:+recent+highlights+and+future+challenges.">Bottom-up synthesis of artificial cells: recent highlights and future challenges.</a> Annu Rev Chem Biomol. Eng. 12 (1), 287-308 (2021).</li><li>Clomburg, J. M., Crumbley, A. M., Gonzalez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Industrial+biomanufacturing:+The+future+of+chemical+production.">Industrial biomanufacturing: The future of chemical production.</a> Science. 355 (6320), (2017).</li><li>Shi, T., Han, P., You, C., Zhang, Y. -. H. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+synthetic+biology+platform+for+emerging+industrial+biomanufacturing:+Bottom-up+pathway+design.">An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-up pathway design.</a> Synth Syst Biotechnol. 3 (3), 186-195 (2018).</li><li>Wang, A., 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=Liver-target+and+glucose-responsive+polymersomes+toward+mimicking+endogenous+insulin+secretion+with+improved+hepatic+glucose+utilization.">Liver-target and glucose-responsive polymersomes toward mimicking endogenous insulin secretion with improved hepatic glucose utilization.</a> Adv Funct Mater. 30 (13), 1910168 (2020).</li><li>Kanter, 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=Cell-free+production+of+scFv+fusion+proteins:+an+efficient+approach+for+personalized+lymphoma+vaccines.">Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.</a> Blood. 109 (8), 3393-3399 (2007).</li><li>Zeltins, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+virus-like+particles:+a+review.">Construction and characterization of virus-like particles: a review.</a> Mol Biotechnol. 53 (1), 92-107 (2013).</li><li>Jain, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology+and+personalized+medicine.">Synthetic biology and personalized medicine.</a> Med Princ Pract. 22 (3), 209-219 (2013).</li><li>Schwille, P., Frohn, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+protein+functions+and+what+they+may+teach+us.">Hidden protein functions and what they may teach us.</a> Trends Cell Biol. 32 (2), 102-109 (2022).</li><li>Sachsenmeier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Industry+5.0-The+relevance+and+implications+of+bionics+and+synthetic+biology.">Industry 5.0-The relevance and implications of bionics and synthetic biology.</a> Engineering. 2 (2), 225-229 (2016).</li><li>Schmidt, D., Jiang, Q. -. X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+the+origin+of+cationic+gating+charges+in+voltage+sensors.">Phospholipids and the origin of cationic gating charges in voltage sensors.</a> Nature. 444 (7120), 775-779 (2006).</li><li>Godoy-Hernandez, A., 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=Rapid+and+highly+stable+membrane+reconstitution+by+LAiR+enables+the+study+of+physiological+integral+membrane+protein+functions.">Rapid and highly stable membrane reconstitution by LAiR enables the study of physiological integral membrane protein functions.</a> ACS Cent Sci. 9 (3), 494-507 (2023).</li><li>Sikkema, H. R., 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=Gating+by+ionic+strength+and+safety+check+by+cyclic-di-AMP+in+the+ABC+transporter+OpuA.">Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA.</a> Sci Adv. 6 (47), 7697 (2020).</li><li>Foucaud, C., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactose+transport+system+of+Streptococcus+thermophilus.+Functional+reconstitution+of+the+protein+and+characterization+of+the+kinetic+mechanism+of+transport.">Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.</a> J Biol Chem. 267 (31), 22087-22094 (1992).</li><li>Yoneda, J. S., Sebinelli, H. G., Itri, R., Ciancaglini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+on+solubilization+and+lipid+reconstitution+of+Na,K-ATPase:+enzyme+kinetic+and+biophysical+characterization.">Overview on solubilization and lipid reconstitution of Na,K-ATPase: enzyme kinetic and biophysical characterization.</a> Biophys Rev. 12 (1), 49-64 (2020).</li><li>Simidjiev, I., 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=Self-assembly+of+large,+ordered+lamellae+from+non-bilayer+lipids+and+integral+membrane+proteins+in+vitro.">Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro.</a> Proc Natl Acad Sci. 97 (4), 1473-1476 (2000).</li><li>Harris, N. J., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+and+stability+of+membrane+transport+proteins+in+vitro.">Folding and stability of membrane transport proteins in vitro.</a> Biochim Biophys Acta BBA - Biomembr. 1818 (4), 1055-1066 (2012).</li><li>Jackson, M. L., Litman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin-egg+phosphatidylcholine+reconstitution+by+an+octyl+glucoside+dilution+procedure.">Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure.</a> Biochim Biophys Acta BBA - Biomembr. 812 (2), 369-376 (1985).</li><li>Geertsma, E. R., Nik Mahmood, N. A. B., Schuurman-Wolters, G. K., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+reconstitution+of+ABC+transporters+and+assays+of+translocator+function.">Membrane reconstitution of ABC transporters and assays of translocator function.</a> Nat Protoc. 3 (2), 256-266 (2008).</li><li>Rigaud, J. -. L., Pitard, B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes:+application+to+energy-transducing+membrane+proteins.">Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins.</a> Biochim Biophys Acta BBA - Bioenerg. 1231 (3), 223-246 (1995).</li><li>Szoka, F., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+preparation+of+liposomes+with+large+internal+aqueous+space+and+high+capture+by+reverse-phase+evaporation.">Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation.</a> Proc Natl Acad Sci. 75 (9), 4194-4198 (1978).</li><li>. Synthetic Organelles for Energy Conservation and Delivery of Building Blocks for Lipid Biosynthesis Available from: <a target="_blank" href="https://www.researchsquare.com/article/rs-3385355/v1">https://www.researchsquare.com/article/rs-3385355/v1</a> (2023)</li><li>Lee, K. 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=Photosynthetic+artificial+organelles+sustain+and+control+ATP-dependent+reactions+in+a+protocellular+system.">Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.</a> Nat Biotechnol. 36 (6), 530-535 (2018).</li><li>M&#233;l&#233;ard, P., Bagatolli, L. A., Pott, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+unilamellar+vesicle+electroformation.">Giant unilamellar vesicle electroformation.</a> Methods in Enzymology. , 161-176 (2009).</li><li>Garten, M., Aimon, S., Bassereau, P., Toombes, G. E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+transmembrane+protein,+the+voltage-gated+ion+channel,+KvAP,+into+giant+unilamellar+vesicles+for+microscopy+and+patch+clamp+studies.">Reconstitution of a transmembrane protein, the voltage-gated ion channel, KvAP, into giant unilamellar vesicles for microscopy and patch clamp studies.</a> J. Vis. Exp. (95), e52281 (2015).</li><li>Doeven, M. K., 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=lateral+mobility+and+function+of+membrane+proteins+incorporated+into+giant+unilamellar+vesicles.">lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles.</a> Biophys J. 88 (2), 1134-1142 (2005).</li><li>Pols, T., 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=A+synthetic+metabolic+network+for+physicochemical+homeostasis.">A synthetic metabolic network for physicochemical homeostasis.</a> Nat Commun. 10 (1), 4239 (2019).</li><li>Bailoni, E., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+recycling+fuels+sustainable+glycerol+3-phosphate+formation+in+synthetic+cells+fed+by+dynamic+dialysis.">ATP recycling fuels sustainable glycerol 3-phosphate formation in synthetic cells fed by dynamic dialysis.</a> ACS Synth Biol. 11 (7), 2348-2360 (2022).</li><li>Van Der Heide, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+osmotic+signal+and+osmosensing+mechanism+of+an+ABC+transport+system+for+glycine+betaine.">On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine.</a> EMBO J. 20 (24), 7022-7032 (2001).</li><li>Van'T Klooster, J. S., 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=Membrane+lipid+requirements+of+the+lysine+transporter+Lyp1+from+Saccharomyces+cerevisiae.">Membrane lipid requirements of the lysine transporter Lyp1 from Saccharomyces cerevisiae.</a> J Mol Biol. 432 (14), 4023-4031 (2020).</li><li>Lou, G., Anderluzzi, G., Woods, S., Roberts, C. W., Perrie, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+microfluidic-based+approach+to+formulate+size-tuneable+large+unilamellar+cationic+liposomes:+Formulation,+cellular+uptake+and+biodistribution+investigations.">A novel microfluidic-based approach to formulate size-tuneable large unilamellar cationic liposomes: Formulation, cellular uptake and biodistribution investigations.</a> Eur J Pharm Biopharm. 143, 51-60 (2019).</li><li>Weiss, 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=Sequential+bottom-up+assembly+of+mechanically+stabilized+synthetic+cells+by+microfluidics.">Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics.</a> Nat Mater. 17 (1), 89-96 (2018).</li><li>Pols, T., Singh, S., Deelman-Driessen, C., Gaastra, B. F., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymology+of+the+pathway+for+ATP+production+by+arginine+breakdown.">Enzymology of the pathway for ATP production by arginine breakdown.</a> FEBS J. 288 (1), 293-309 (2021).</li><li>Yandrapalli, N., Robinson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+capacity+microfluidic+trapping+of+giant+vesicles+for+high-throughput+membrane+studies.">Ultra-high capacity microfluidic trapping of giant vesicles for high-throughput membrane studies.</a> Lab Chip. 19 (4), 626-633 (2019).</li><li>Elias, 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=Microfluidic+characterization+of+biomimetic+membrane+mechanics+with+an+on-chip+micropipette.">Microfluidic characterization of biomimetic membrane mechanics with an on-chip micropipette.</a> Micro Nano Eng. 8, 100064 (2020).</li><li>Robinson, T., Kuhn, P., Eyer, K., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+trapping+of+giant+unilamellar+vesicles+to+study+transport+through+a+membrane+pore.">Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore.</a> Biomicrofluidics. 7 (4), 044105 (2013).</li><li>Cooper, A., Girish, V., Subramaniam, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+Pressure+Enables+High-Yield+Assembly+of+Giant+Vesicles+in+Solutions+of+Physiological+Ionic+Strengths.">Osmotic Pressure Enables High-Yield Assembly of Giant Vesicles in Solutions of Physiological Ionic Strengths.</a> Langmuir. 39 (15), 5579-5590 (2023).</li><li>Tantama, M., Mart&#237;nez-Fran&#231;ois, J. R., Mongeon, R., Yellen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+energy+status+in+live+cells+with+a+fluorescent+biosensor+of+the+intracellular+ATP-to-ADP+ratio.">Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.</a> Nat Commun. 4 (1), 2550 (2013).</li><li>Setyawati, I., 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=In+vitro+reconstitution+of+dynamically+interacting+integral+membrane+subunits+of+energy-coupling+factor+transporters.">In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters.</a> eLife. 9, e64389 (2020).</li><li>Oropeza-Guzman, E., R&#237;os-Ram&#237;rez, M., Ruiz-Su&#225;rez, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveraging+the+coffee+ring+effect+for+a+defect-free+electroformation+of+giant+unilamellar+vesicles.">Leveraging the coffee ring effect for a defect-free electroformation of giant unilamellar vesicles.</a> Langmuir. 35 (50), 16528-16535 (2019).</li><li>Estes, D. J., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroformation+of+giant+liposomes+from+spin-coated+films+of+lipids.">Electroformation of giant liposomes from spin-coated films of lipids.</a> Colloids Surf B Biointerfaces. 42 (2), 115-123 (2005).</li></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="Figure 1" class="xfigimg" src="/files/ftp_upload/66627/66627fig01v2.jpg" /> 
Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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-&#946;-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="Figure 2" class="xfigimg" src="/files/ftp_upload/66627/66627fig02v2.jpg" /> 
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.remotexs.ntu.edu.sg/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="Figure 3" class="xfigimg" src="/files/ftp_upload/66627/66627fig03v2.jpg" /> 
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 &#181;M. (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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

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="Figure 5" class="xfigimg" src="/files/ftp_upload/66627/66627fig05v2.jpg" /> 
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.remotexs.ntu.edu.sg/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&#160;to more than 50 &#956;m.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66627/66627fig06v2.jpg" /> 
Figure 6: DIC images of proteoGUVs formed by gel-assisted swelling. Scale bar = 25 &#181;m. Abbreviations: DIC = differential interference contrast; GUVs = giant-unilammellar vesicles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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 &#946;-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="Figure 7" class="xfigimg" src="/files/ftp_upload/66627/66627fig07.jpg" /> 
Figure 7: Trapping of vesicles with the microfluidic chip. (A)&#160;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 &#62;500 nm. The brightfield image allows for visualization of the vesicles without fluorescence. Scale bar = 25 &#181;m. Abbreviation: GUVs = giant-unilamellar vesicles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66627/66627fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Author Spotlight: Tackling Challenges in Synthetic Cell Engineering at JoVE.com

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

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.

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, ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

980 Views.  University of Groningen. In Nederland wil het BaSyC-consortium synthetische cellen bouwen uit moleculaire componenten. Het BaSyC-consortium bestaat uit onderzoeksgroepen variërend van moleculaire biologie tot scheikunde, natuurkunde en computationele wetenschappen. Het doel van dit consortium is om een zelfreplicerend systeem te creëren dat in staat is tot autonome groei, om informatie te verzenden en te verdelen.En we richten ons momenteel op het bouwen van moleculaire netwerken die worden geïntegreerd in ...

Video: Auteur in de schijnwerpers: uitdagingen aanpakken in synthetische celtechniek

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

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

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

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.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

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.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

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

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

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

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Immunology and Infection

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

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.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

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

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