APL acknowledges support from the National Science Foundation (EF1935265), the National Institutes of Health (R01-EB030031 and R21-AR080363), and the Army Research Office (80523-BB)

The reagents and equipment utilized for this study are provided in the Table of Materials.
1. Bulk CFE reactions in the presence of small unilamellar vesicles (SUVs)
<ol>
	<li>SUV preparation 
	NOTE: This step needs to be performed in a fume hood following the safety instructions for working with chloroform.
	<ol>
		<li>Prepare 5 mM 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) SUVs in a glass vial by transferring 76 &#181;L of 25 mg/mL POPC stock solution dissolved in chloroform.</li>
		<li>While gently rotating the glass vial, blow a gentle stream of argon into the vial to form a film of dried lipids at the bottom. Next, transfer the glass vial to a desiccator with its cap loosely screwed to evaporate excess chloroform.</li>
		<li>Keep the glass vial in the desiccator for 1 h. Then, add 0.5 mL ultra-pure deionized water to dissolve the lipid film and vortex for approximately 2 min.</li>
		<li>Set up a mini-extrusion apparatus by soaking two filter supports in deionized water and placing them on each of the internal membrane supports. Then, soak a 100 nm polycarbonate filter and place it in between the two internal membrane supports held together by the extruder outer casing and the retainer nut. Place this setup in the extruder stand.</li>
		<li>Flush two 1 mL gas-tight syringes 3 times with ultra-pure deionized water.</li>
		<li>Load the sample of lipid-water mix into one of the 1 mL gas-tight syringes and place it in one end of the mini extruder using the swing arm clips to hold the syringe in place. Insert the second syringe into the other end of the mini-extruder and make sure it is fully depressed. 
		NOTE: When loading the syringe with the lipid-water mix, ensure there is no air in the syringe before passing it through the mini-extruder.</li>
		<li>Gently pass the lipid-water mix from the original syringe to the empty syringe through the mini-extruder apparatus. Repeat this step 11 times to form SUVs. Transfer the SUVs to a 1.5 mL microcentrifuge tube. 
		NOTE: The SUV solution can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>CFE reaction assembly
	<ol>
		<li>Assemble the CFE reaction by following the cell-free expression protocol provided by the manufacturer with slight modifications detailed in the following.</li>
		<li>Mix 10 &#181;L of Solution 1 (containing amino acids, NTPs, tRNAs and substrates for enzymes, and necessary buffer), 1 &#181;L of Solution 2 (proteins in 20% glycerol), 2 &#181;L of Solution 3 (ribosome (20 &#181;M)), appropriate amount of the DNA encoding for soluble sfGFP-sfCherry(1-10)27&#160;(referred to as soluble sfGFP hereafter), WT-GluR0-sfGFP, or PRSP-GluR0-sfGFP (10-60 ng/1000 base pairs), 1 &#181;L murine RNAse inhibitor, and 4 &#181;L of 5 mM SUV solution for membrane proteins or 4 &#181;L water for soluble proteins.</li>
		<li>Bring the reaction&#39;s final volume to 20 &#181;L by adding ultra-pure deionized water. 
		NOTE: When assembling the CFE system, all components must be kept on ice. All materials are temperature-sensitive and can degrade if they reach room temperature.</li>
	</ol>
	</li>
	<li>CFE reaction incubation and monitoring
	<ol>
		<li>Transfer the CFE solution to a 96-well conical V-bottom plate. To prevent evaporation during the course of the reaction, cover the plate using a sealing film.</li>
		<li>Incubate the plate at 37 &#176;C in a plate reader for 4-5 h while monitoring the CFE reaction by measuring the GFP signal with a gain of 100 at 488 nm/528 nm excitation/emission wavelengths every 2 min.</li>
	</ol>
	</li>
</ol>
2. CFE reactions encapsulated in GUVs
<ol>
	<li>Preparation of GUV outer buffer solution
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, mix 1.5 &#181;L of 1 M spermidine, 37.5 &#181;L of 100 mM ATP, 25 &#181;L of 100 mM GTP, 12.5 &#181;L of 100 mM CTP, 12.5 &#181;L of 100 mM UTP, 25 &#181;L of 1 M creatine phosphate, 18 &#181;L of 1 M magnesium acetate, 93.33 &#181;L of 3 M potassium glutamate, 50 &#181;L of 1 M HEPES KOH (pH 7.4), 1.15 &#181;L of 332 mM folinic acid, 100 &#181;L of 2 M glucose, and 50 &#181;L from stock solution of 6 mM mixture of each of the 20 amino acids (prepared by following the protocol described by Sun et al.28).</li>
		<li>Bring the solution&#39;s final volume to 1 mL by adding ultra-purified deionized water. 
		NOTE: All components must be kept on ice. Add the amino acid mixture at the end to avoid amino acid depletion28. The solution can be aliquoted in 330 &#181;L aliquots and stored at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Preparation of the lipid-in-oil mixture 
	NOTE: this step needs to be performed in a fume hood following the safety instructions for working with chloroform.
	<ol>
		<li>In a fume hood, mix 17.3 &#181;L of 25 mg/mL POPC stock solution and 1.08 &#181;L of 50 mg/mL poly(butadiene)-b-poly(ethylene oxide) (PEO-b-PBD) copolymer in a 15 mL glass vial. 
		NOTE: The final lipid-in-oil solution contains 0.5 mM lipid with 95% and 5% POPC and PEO-b-PBD, respectively. PEO-b-PBD was used to enhance membrane stability during protein expression but was kept at a low molar ratio to reduce the copolymer&#39;s tendency to aggregate into micelles separate from lipid molecules29,30.</li>
		<li>Carefully blow a gentle stream of argon gas into the glass vial while rotating the vial to evaporate the chloroform.</li>
		<li>Pipette 1.2 mL of light mineral oil into the 15 mL glass vial containing the dried lipids.</li>
		<li>Mix the lipids and oil by vortexing at maximum speed for 10-20 s. The dissolved lipid-copolymer mix will look cloudy.</li>
		<li>To ensure that all possible lipid aggregates in the oil are fully dissolved and dispersed throughout the oil, place the glass vial in an oven at around 50 &#176;C for 20 min before vortexing for an additional 10-20 s at maximum speed.</li>
	</ol>
	</li>
	<li>CFE reaction assembly and encapsulation (Figure 1 summarizes the following steps).
	<ol>
		<li>Prepare 300 &#181;L of the GUV outer solution in a 1.5 mL microcentrifuge tube by mixing 270 &#181;L of CFE outer buffer solution prepared in step 2.1, 15 &#181;L of 5 M NaCl, and 15 &#181;L of 4.5 M KCl. 
		NOTE: At this step, add 0.45 &#181;L of 1 M 1,4-dithiothreitol (DTT) to the GUV outer solution. The purpose of adding NaCl and KCl is to adjust the outer solution&#39;s osmolality to match it with the inner CFE solution. The exact volume of NaCl and KCl depends on the desired osmolality adjustment. One can add either NaCl or KCl or both to adjust the osmolality.</li>
		<li>Gently pipette 300 &#181;L of lipid-in-oil mixture on top of the GUV outer solution. 
		NOTE: When adding the lipid-oil mix, it is important that the oil does not mix with the aqueous outer solution. After the addition, there should be a visible interface between the lipid-in-oil mixture and the GUV outer solution.</li>
		<li>Incubate the oil-water interface at room temperature for 2 h to allow the lipid monolayer to form and stabilize at the interface.</li>
		<li>Meanwhile, follow section 1.2.1 to assemble a CFE reaction containing the plasmid DNA encoding the membrane protein variants or soluble GFP. Replace the 4 &#181;L of 5 mM SUV solution or water with 4 &#181;L of 1 M sucrose. This reaction will be the GUV inner solution. 
		NOTE: An osmometer was used to measure the osmolality of the inner and outer solutions. The osmolality of the outer solution was then adjusted accordingly by the addition of NaCl or water. The osmolality of the CFE reaction is typically around 1600 mOsm/Kg. Adding sucrose to the reaction increases the inner solution density, thus allowing the vesicles to travel across the oil-water interface during the centrifugation step. An alternative to sucrose is the Opti-Prep density gradient solution.</li>
		<li>Add 600 &#181;L of lipid-oil mixture to the microcentrifuge tube containing the CFE reaction and pipette up and down vigorously for ~1 min to emulsify the reaction in lipid-in-oil solution and form the lipid monolayer around synthetic cells. 
		NOTE: The final solution should not have any bubbles and should look opaque.</li>
		<li>Gently pipette the inner solution emulsion on top of the oil layer in the 1.5 mL microcentrifuge tube where the oil-water interface was set up. 
		NOTE: Be careful not to disturb or destabilize the interface.</li>
		<li>Centrifuge for 10 min at 2,000 x g at 4 &#176;C. 
		NOTE: The centrifugation speed was optimized for this protocol. Adir et al.31 reported a different centrifugation speed.</li>
		<li>Once the centrifugation is over, carefully remove the excess oil and outer solution from the microcentrifuge tube using a pipettor. Remove the outer solution until the remaining volume is around 100 &#181;L. 
		NOTE: A pellet of GUVs is usually visible at the bottom of the microcentrifuge tube. However, a lack of a visible pellet does not necessarily mean no GUV yield. Instead of using a pipettor, the excess oil on top of the outer solution can be removed by aspiration. It is critical to ensure the lipid-in-oil solution is completely removed. Oil contamination in GUV solution can cause low-quality images.</li>
		<li>Resuspend the GUV pellet in the remaining 100 &#181;L solution by gently pipetting up and down. Next, transfer the GUV solution to a clean 96 well clear flat bottom plate to incubate.</li>
	</ol>
	</li>
</ol>
3. Encapsulated CFE reaction incubation and imaging
<ol>
	<li>Cover the plate using a sealing film to prevent evaporation. Incubate the plate at 37 &#176;C for 5-6 h. One can use a plate reader and follow step 1.3.2 to prepare the plate reader for the incubation step.</li>
	<li>Once the incubation is over, place the 96 well plate on the imaging stage of an inverted microscope equipped with an EMCCD camera (or a sCMOS camera), DAQ-MX controlled laser (or an integrated laser combiner system), and a CSU-X1 spinning disk confocal (or a laser scanning confocal). Focus on any ROI containing GUVs and capture images at an excitation wavelength of 488 nm using a Plan-Apochromat 60 x/1.4 NA objective.</li>
	<li>Save images of GUVs in .tiff format.</li>
	<li>Open the images in an image processing software (e.g., ImageJ or Fiji). Open the Brightness/Contrast setting panel. Adjust the brightness and contrast to appropriate settings that make fluorescent proteins visible.</li>
	<li>If the goal is to compare the signal intensity of different expressed proteins, first stack individual images of GUVs containing different proteins using Images to Stack panel located under Image &#62; Stacks submenu. Then, adjust the brightness and contrast of all images using the Brightness/Contrast panel.</li>
</ol>

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

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R., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+polymersomes:+a+comparative+review+towards+cell+mimicking.">Liposomes and polymersomes: a comparative review towards cell mimicking.</a> Chem Soc Rev. 47 (23), 8572-8610 (2018).</li><li>Tsumoto, K., Hayashi, Y., Tabata, J., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reverse-phase+method+revisited:+Rapid+high-yield+preparation+of+giant+unilamellar+vesicles+(GUVs)+using+emulsification+followed+by+centrifugation.">A reverse-phase method revisited: Rapid high-yield preparation of giant unilamellar vesicles (GUVs) using emulsification followed by centrifugation.</a> Colloids Surf A: Physicochem Eng Asp. 546, 74-82 (2018).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> J Am Chem Soc. 137 (33), 10767-10776 (2015).</li><li>Bailoni, E., 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=Minimal+out-of-equilibrium+metabolism+for+synthetic+cells:+A+membrane+perspective.">Minimal out-of-equilibrium metabolism for synthetic cells: A membrane perspective.</a> ACS Synth Biol. 12 (4), 922-946 (2023).</li></ol>

The authors declare no conflicts of interest.

Virtually any cellular process that depends on the transfer of molecules or information across the cell membrane, like cell signaling or cell excitation, requires membrane proteins. Thus, the reconstitution of membrane proteins has become the main bottleneck in realizing various synthetic cell designs for different applications. Traditional detergent-mediated reconstitution of membrane proteins in biological membranes requires GUV generation methods such as gentle swelling or electroformation. Swelling approaches usually...

Bottom-up synthetic biology has gained increasing interest over the past decade as an emerging field with numerous potential applications in bioengineering, drug delivery, and regenerative medicine1,2. The development of synthetic cells as a cornerstone of bottom-up synthetic biology, in particular, has attracted a wide range of scientific communities due to the promising applications of synthetic cells as well as their cell-like physical and biochemical properties that facilitate in vitro biophysical studies3,4,5,6. Synthetic cells are often engineered in cell-sized giant unilamellar vesicles (GUVs) in which different biological processes are recreated. Reconstitution of cell cytoskeleton7,8, light-dependent energy regeneration9, cellular communication10,11, and biosensing12 are examples of efforts made to reconstruct cell-like behaviors in synthetic cells.
While some cellular processes rely on soluble proteins, many characteristics of natural cells, such as sensing and communication, often utilize membrane proteins, including ion channels, receptors, and transporters. A major challenge in synthetic cell development is the reconstitution of membrane proteins. Although traditional methods of membrane protein reconstitution in lipid bilayers rely on detergent-mediated purification, such methods are laborious, ineffective for proteins that are toxic to the expression host, or are often not suited for membrane protein reconstitution in GUVs13.
An alternative method for protein expression is cell-free expression (CFE) systems. CFE systems have been a powerful tool in synthetic biology that allows in vitro expression of various proteins using either cell lysate or purified transcription-translation machinery14. CFE systems can also be encapsulated in GUVs, thus allowing compartmentalized protein synthesis reactions that can be programmed for various applications, such as the creation of light-harvesting synthetic cells9 or mechanosensitive biosensors15,16. Analogous to recombinant protein expression methods, membrane protein expression is challenging in CFE systems17. Aggregation, misfolding, and lack of post-translational modification in CFE systems are major bottlenecks that hinder successful membrane protein synthesis using CFE systems. The difficulty of bottom-up membrane protein reconstitution using CFE systems is due in part to the absence of a complex membrane protein biogenesis pathway that relies on signal peptides, signal recognition particles, translocons, and chaperoning molecules. However, recently, multiple studies have suggested that the presence of membranous structures such as microsomes or liposomes during translation promotes successful membrane protein expression18,19,20,21. Additionally, Eaglesfield et al. and Steink&#252;her et al. have found that the inclusion of specific hydrophobic domains known as signal peptides in the N-terminus of the membrane protein can significantly improve its expression22,23. Altogether, these studies suggest that the challenge of membrane protein reconstitution in synthetic cells can be overcome if the protein translation occurs in the presence of the GUV membrane and if proper N-terminal signal peptide is utilized.
Here, a protocol for encapsulation of the protein synthesis using recombinant elements (PURE) CFE reactions for membrane protein reconstitution in GUVs is presented. Bacterial glutamate receptor24 (GluR0) is selected as the model membrane protein, and the effect of its N-terminal signal peptide on its membrane reconstitution is studied. The effect of proteorhodopsin signal peptide, which was shown to improve membrane protein reconstitution efficiency by Eaglesfield et al.22, is investigated by constructing a mutated variant of GluR0 denoted as PRSP-GluR0 and its expression and membrane localization with wild-type GluR0 (referred to as WT-GluR0 hereafter) that harbors its native signal peptide is compared. This protocol is based on the inverted emulsion method25 with modifications that make it robust for CFE encapsulation. In the presented method, the CFE reactions are first emulsified using a lipid-in-oil solution that generates micron-sized droplets that contain the CFE system and are stabilized by the lipid monolayer. The emulsion droplets are then layered on top of an oil-water interface that is saturated with another lipid monolayer. The emulsion droplets are then forced to travel across the oil-water interface via centrifugal force. Through this process, the droplets obtain another monolayer, thus generating a bilayer lipid vesicle. The GUVs containing the CFE reaction are then incubated, during which the membrane protein is expressed and incorporated into the GUV membrane. Although this protocol is specified for cell-free expression of GluR0, it can be used for cell-free synthesis of other membrane proteins or different synthetic cell applications such as cytoskeleton reconstitution or membrane fusion studies26.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 nm polycarbonate filter</td>
			<td>STERLITECH</td>
			<td>1270193</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Clear Bottom Plate</td>
			<td>ThermoFisher Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy H1M Hybrid Multi-Mode Reader</td>
			<td>Agilent</td>
			<td>11-120-533</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Millipore Sigma</td>
			<td>10621714001</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1 Confocal Scanner Unit</td>
			<td>Yokogawa</td>
			<td>CSU-X1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium (Optiprep)</td>
			<td>Millipore Sigma</td>
			<td>D1556</td>
			<td>Optional to switch with sucrose in inner solution</td>
		</tr>
		<tr>
			<td>Filter supports</td>
			<td>Avanti</td>
			<td>610014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand microtubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Millipore Sigma</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>158968</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>iXon X3 camera&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid potassium salt monohydrate</td>
			<td>Millipore Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Millipore Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate&#160;</td>
			<td>Millipore Sigma</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder kit (including syringe holder and extruder stand)</td>
			<td>Avanti</td>
			<td>610020</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX81 Inverted Microscope&#160;</td>
			<td>Olympus</td>
			<td>IX21</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus PlanApo N 60x Oil Microscope Objective&#160;</td>
			<td>Olympus</td>
			<td>1-U2B933&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-b-PBD</td>
			<td>Polymer Source</td>
			<td>P41745-BdEO</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-PRSP-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-sfGFP-sfCherry(1-10) plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-WT-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC lipid in chloroform&#160;</td>
			<td>Avanti</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>PUREfrex 2.0</td>
			<td>Cosmo Bio USA</td>
			<td>GFK-PF201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleotide Solution Set</td>
			<td>New England BioLabs</td>
			<td>N0450</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor, Murine</td>
			<td>New England BioLabs</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>Biotechrabbit</td>
			<td>BR1401801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Millipore Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>VAPRO Vapor Pressure Osmometer Model 5600</td>
			<td>ELITechGroup</td>
			<td>VAPRO 5600</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Prior to encapsulation of the CFE reactions, two variants of GluR0-sfGFP harboring native and proteorhodopsin signal peptides (signal peptide sequences are presented in Supplementary&#160;Table 1), and the soluble sfGFP were individually expressed in bulk reactions, and their expression was monitored by detecting the sfGFP signal using a plate reader (Figure 2A). Membrane proteins were expressed in the absence or presence of 100 nm SUVs. Additionally, using ...

Watch this Scientific Journal Video about Membrane Protein Reconstitution in Synthetic Cells at JoVE.com

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.

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 ...

reconstitution-bacterial-glutamate-receptor-channel-encapsulation

Research

JoVE Journal

Bioengineering

979 Views.  University of Michigan, Ann Arbor. 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.

Video: Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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 ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...