Name: Author Spotlight: Tackling Challenges in Synthetic Cell Engineering
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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).

1. General preparation
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	<li>Chemicals
	<ol>
		<li>Dissolve lipids (in powder form) to 25 mg/mL in CHCl3 for making preformed liposomes. 
		NOTE: It is preferable to prepare fresh lipid stocks, but the stock solutions can also be stored at -20 &#176;C for a few weeks. Working with lipids in powder form is more accurate than using lipids already solubilized in CHCl3. CHCl3 should be handled using glass pipettes and/or syringes and stored in glass containers as C...

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. 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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-phosph...

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/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/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/files/ftp_upload/66627/66627fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BioBeads</td>
			<td>BioRad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>CHCl3</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&#160;</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 &#181;L</td>
			<td>Hamilton</td>
			<td>7656-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 1000 &#181;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>K2HPO4</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>KH2PO4</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>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic chip</td>
			<td>Homemade&#160;</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&#39;&#39; 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&#39;&#39; 1.2 x 40 mm</td>
		</tr>
		<tr>
			<td>Ni2+ 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 &#181;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&#160;</td>
			<td>GE Healthcare</td>
			<td>17-0050-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics Sonics &#38; 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 &#181;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&#160;</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>&#946;-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.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

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

In the Netherlands, the BaSyC consortium aims to construct synthetic cells from molecular components. The BaSyC consortium is composed of research groups ranging from molecular biology to chemistry, physics, and computational sciences. The goal of this consortium is to create a self-replicating system capable of autonomous growth to transmit information and to divide.And we currently focus on the building of molecular networks that are integrated towards more complex molecular systems. Recent developments in synthetic biology include the engineering of modules for cellular functions, integrating them into complex networks for applications in diagnostics, therapy, synthetic immunology, and adaptive materials. This progress spans groups in Europe, America, and Asia, advancing both top-down and bottom-up approaches to synthetic cell construction.Current technologies in our field include the engineering and evolutionary methods to construct and optimize metabolic networks. We use AI for molecular selection and advanced biochemical and biophysical techniques for component isolation and network formation. We also employ membrane reconstitution, physical encapsulation, and visualization via fluorescent spectroscopy and microscopy.With the integration of modules, the number of membrane proteins and enzymes increase enormously, which poses constraints on our reconstitution and encapsulation technologies. Stochastic encapsulation becomes an issue with sub-micron size vesicles, and the efficiency of forming micrometer-size vesicles decreases when multiple membrane proteins are reconstituted in the lipid membrane. In Groningen, we have developed metabolic networks for ATP productions and maintaining an out-of-equilibrium state through substrate feeding and product export.Additionally, we linked ATP recycling to the synthesis and excretion of lipid precursors, enabling secondary vesicles to produce lipids, which is a significant advancement in our field.

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...

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

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-outofequilibrium-metabolic-networks-nano-micrometersized