The authors acknowledge the Armenise-Harvard Foundation, the Marie-Curie Trentino COFUND (ACS), the Autonomous Province of Trento (Ecomm), and CIBIO for funding.

1. Preparing the DNA Template
<ol>
	<li>Purify plasmid from a standard laboratory strain of E. coli, such as E. coli DH5a or Nova Blue with a commercial kit. Alternatively, a linear PCR product can be similarly purified with a commercial kit. Elute the DNA with H2O only.</li>
	<li>Phenol-chloroform extract the DNA solution26.</li>
	<li>Determine the DNA concentration and purity, e.g. by UV absorbance or other suitable methods. It is important to use highly pure DNA for efficient transcription-translation.</li>
</ol>
2. Preparing the Thin Lipid Film
<ol>
	<li>Weigh the dry lipid powder and dissolve in solvent. For 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), stock solutions are prepared in chloroform at a concentration of 40 mg/ml. 
	Note: Organic solvents must always be handled with glass pipettes and bottles. In other words, plastic must never be used. Store stock solutions in air tight, solvent resistant amber glass bottles at -20 &#176;C, preferably under argon.</li>
	<li>Aliquot 12 &#956;mol POPC (220 &#956;l&#160;of 40 mg/ml&#160;stock solution) into a 5 ml&#160;round bottom flask.</li>
	<li>Evaporate the solvent with a rotary evaporator (Figure 1) to generate the thin lipid film.
	<ol>
		<li>Securely attach the round bottom flask to the distillation tube with a circular clip and start the rotation of the flask.</li>
		<li>Initiate the circulation of water through the condenser coil. Using a heat bath is not necessary, because chloroform has a low boiling point of 61.2 &#176;C at normal atmospheric pressure.</li>
		<li>Ensure that the system is open to the atmosphere by checking that the stopcock is open. Turn on the vacuum pump and slowly close the stopcock to apply the vacuum to the system.</li>
		<li>A thin lipid film is deposited on the walls of the round bottom flask during rotary evaporation forming an opaque film that is visible by eye. Let the rotary evaporation continue for 0.5-2 hr. To stop the process, slowly release the vacuum pressure, stop the rotation, and remove the round bottom flask.</li>
	</ol>
	</li>
</ol>
3. Lipid Resuspension and Vesicle Homogenization
<ol>
	<li>Add 1 ml&#160;18.2 M&#937; H2O to the thin lipid film directly in the round bottom flask. Vigorously vortex the solution at maximum speed, approximately 3,200 rpm, until the lipid film detaches from the glass, which is observable by eye.</li>
	<li>Transfer the lipid dispersion into a 2 ml&#160;microcentrifuge tube.</li>
	<li>Set-up a ring stand to hold a homogenizer with a 5 mm tip. Place a microcentrifuge stand below the homogenizer to securely hold the lipid dispersion. Rinse the dispersing element of the homogenizer by immersing the tip in 18.2 M&#937; H2O and running for a few seconds.</li>
	<li>Place the dispersing element directly into the lipid dispersion ensuring that the tip does not touch the bottom of the microcentrifuge tube. Homogenize for 1 min at power level 4 or 14,000 rpm.</li>
</ol>
4. Vesicle Extrusion and Lyophilization
<ol>
	<li>Assemble the parts of the mini-extruder (Figure 2), which consists of an outer casing and a retainer nut that houses two Teflon internal membrane supports, two O-rings, and one Teflon bearing.
	<ol>
		<li>Place the two O-rings into the grooves of the internal membrane supports. Prewet two filters and one 400 nm membrane. The filters will be placed against the Teflon inside of the O-rings with the membrane between them.</li>
		<li>Place the membrane supports into the extruder outer casing with the membrane surrounded by two filters in between the O-rings. Place the Teflon bearing inside the casing and attach the retainer nut and tighten by hand.</li>
	</ol>
	</li>
	<li>Rinse two syringes and fill one with 18.2 M&#937; water. Insert the syringe needles into the small holes in the Teflon on either end of the extruder assembly. The needles should slide in easily; do not force the needles. Secure the extruder with the syringes into the extruder housing and fasten.</li>
	<li>Pass the water through the extruder by slowly pushing the water out of one syringe and into the other. This represents one passage. Repeat for a total of three passages ensuring that there are no leaks present. Remove the syringe of water and dispose of the water.</li>
	<li>Fill one syringe with the sample, attach to the extruder, and slowly pass the vesicle solution through the membrane as described above in step 4.3. Repeat 10x&#160;for a total of 11 passages. As the extrusion process proceeds, the sample will become less turbid and easier to push across the membrane. A sudden decrease in resistance, however, usually indicates a rupturing of the membrane.</li>
	<li>Transfer the extruded vesicle solution and make 40 &#956;l&#160;aliquots of the vesicles into microcentrifuge tubes. Flash freeze each aliquot in either dry ice or in liquid nitrogen.</li>
	<li>Lyophilize each aliquot with a centrifugal evaporator overnight at 30 &#176;C. Store the lyophilized empty vesicles at -20 &#176;C.</li>
</ol>
5. Encapsulating Transcription-translation Machinery
<ol>
	<li>Mix the components of the transcription-translation reaction and add 20 units of RNase inhibitor. Incubate on ice.</li>
	<li>Add the DNA template. For a control reaction, 250 ng of a plasmid encoding mVenus or a similar fluorescent protein behind a T7 transcriptional promoter and a strong E. coli ribosome binding site is recommended.</li>
	<li>Bring the final volume to 25 &#956;l&#160;with RNase-free water.</li>
	<li>Hydrate an aliquot of lyophilized vesicles (from step 4.6) with 10 &#956;l&#160;of the reaction assembled in step 5.3. Briefly vortex the mixture until the vesicles are resuspended. This should take less than 30 sec.</li>
	<li>Incubate the reaction on ice for 30 min to allow the vesicles to swell.</li>
	<li>Dilute the vesicle mixture 20-fold to a final volume of 30 &#956;l&#160;by adding 1.5 &#956;l&#160;of vesicles into 27.0 &#956;l&#160;of 50 mM Tris-HCl, 50 mM NaCl, pH 7.4 and 1.5 &#956;l&#160;of 20.2 mg/ml&#160;Proteinase K. DNase and RNase can also be added at this point as an alternative to proteinase K to degrade extravesicular material.</li>
	<li>Incubate for at least 2.5 hr at 37 &#176;C.</li>
</ol>
6. Microscopy
<ol>
	<li>Examine the vesicles and the progress of fluorescent protein production at different time points. The vesicles will have a larger diameter than the 400 nm pore-size of the membrane used for vesicle extrusion.
	<ol>
		<li>Prepare a sample chamber by placing a 20 x 5 mm silicon spacer onto a standard microscope slide. Pipette 10 &#956;l&#160;of vesicles into the sample chamber. Place a siliconized glass cover slip over the chamber.</li>
		<li>Observe the vesicles with a 63X oil-dispersion or similar objective by bright field and fluorescence microscopy using the appropriate filter set for the exploited fluorescent protein.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although cell-free synthetic biology is still in its infancy, advances have laid a foundation from which increasingly complex cell-like systems can be made. The reconstitution of transcription-translation machinery from fully defined components9&#160;inside of vesicles28&#160;was particularly significant in facilitating later efforts in constructing environmentally responsive artificial cells17,18.&#160;Similarly, artificial cell studies have been used to probe evolutionary processes4,29,30, the mechanistic details of RNA and protein synthesis22,31, the influences of metabolic load32,33,&#160;and the assembly of viral particles34. Importantly, enough knowledge now exists that basic cellular function can be reconstituted inside of vesicles in the laboratory following these previous reports and the protocols described herein.
In addition to being easy, the described encapsulation procedure has several benefits. For example, many empty, lyophilized vesicle aliquots can be made in advance and stored at -20 &#176;C for later use. The protocol does not subject biological molecules to organic solvents, drastic temperature changes, or long periods of dialysis. We expect that the gentleness of the procedure will facilitate the incorporation of additional components as needed. We also have not observed adverse effects to changing the lipid composition of the membrane on encapsulation or transcription-translation efficiency. Therefore, lipids more amenable to the incorporation of membrane proteins, specific morphologies, or visualization could conceivably be exploited.
The major limitation of the described method is that the resulting vesicles are not homogenous in size or lamellarity. For many applications, these difficulties do not interfere with the interpretation of data. However, if needed, additional steps can be incorporated to narrow the size distribution and decrease the layers of membranes, such as further rounds of extrusion after encapsulation, freeze-thawing, or dialysis35.&#160;Undoubtedly better methods that circumvent these and other problems will be developed. Until then, we find the protocol described here to be well suited for the construction of cellular mimics.

Cell-free, in vitro transcription-translation reactions and the generation of vesicles from synthetic lipids are nothing new. However, combining the two into a cellular mimic is significantly more challenging1-6.&#160;E. coli cell extracts with or without T7 RNA polymerase can be used as a source of transcription-translation machinery7,8. Cell extracts benefit from the presence of additional cellular components that can facilitate protein expression and folding. Alternatively, a mix of individually purified RNA and protein molecules, i.e. the PURE system9, can be used to mediate intravesicular protein synthesis4,10-14. The PURE system allows for the construction of fully defined cellular mimics and does not suffer from the nuclease activity found in cell extracts. Practically, this means that much less DNA template is required, thereby facilitating processes with low encapsulation efficiency11. Although less frequently used, cellular mimics can be built with cell extracts derived from eukaryotic cells15.&#160;Thus far, genetically encoded encapsulated cascades and cellular mimics that sense the environment have been reported16-18.
The simplest way to monitor transcription-translation reactions is to measure the fluorescence or luminescence of genetically encoded elements. Typically, firefly luciferase19&#160;or GFP are used, although in vitro reactions are frequently measured by radiolabeling. Fluorescence detection additionally allows for the monitoring of populations of vesicles20,21 through cytometry based methods, thereby offering some insight into the stochastic nature of biological-like processes. These monitoring methods have been used to define a small set of design rules and a library of parts from which to build from, including a collection of fluorescent proteins that are compatible with in vitro transcription-translation22,&#160;the influence of genetic organization on expression22,&#160;the activity of sigma factors16, and the efficiency of transcriptional terminators23. Nevertheless, there remains much that needs to be done to increase the capability of building predictable in vitro, genetically encoded devices.
There are many methods available to make vesicles. The most common methods depend upon the generation of a thin lipid film on a glass surface followed by resuspension in aqueous solution24.&#160;If the aqueous solution contains transcription-translation machinery, for example, then a fraction of the vesicles formed would contain the necessary components for protein production. However, the encapsulation efficiency of such methods is low, meaning that only a small percentage of vesicles are active. Many of the alternative methods characterized by much higher encapsulation efficiency exploit the conversion of water-in-oil emulsion droplets to vesicles. While it is likely that such methods will be commonplace in the future, currently these methods suffer from the need of specialized equipment and give vesicles with altered membrane compositions25. A distinct advantage of water-in-oil to vesicle methods is the potential to control membrane lamellarity. The method described herein is based on the thin lipid film protocol described by the Yomo laboratory11&#160;with slight modifications including an additional homogenization step. This method is easy, cheap, and gives robust vesicles well suited for the encapsulation of transcription-translation machinery.

<table><tbody><tr><td>Quick Spin Mini-prep kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Spectrometer</td><td>NanoDrop 1000</td><td>NDB767ND</td><td></td></tr><tr><td>POPC</td><td>Avanti Polar Lipids</td><td>770557</td><td>MW 760 g/mol&lt;br/&gt; Transition Temp -2 &amp;deg;C&lt;br/&gt; CAS# 26853-31-6</td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>459836</td><td>Anhydrous, &gt;99.5%</td></tr><tr><td>Phenol-choloroform-isoamyl alcohol 25:24:1, for molecular biology use</td><td>Sigma Aldrich</td><td>P3803-100mL</td><td>Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA</td></tr><tr><td>Chloroform</td><td>Biotech Grade Fluka</td><td>496189-1L</td><td>Contain ethanol at 0.5-1.0% v/v as stabilizer</td></tr><tr><td>Brown amber glass bottles</td><td>VWR</td><td>89043-518</td><td>55X 48 mm</td></tr><tr><td>Rotary evaporator</td><td>Buchi Rotovapor R-210/Sigma</td><td>Z563846EU-1EA</td><td>With jack and water bath, 29/32 joint 240 V</td></tr><tr><td>Analog vortex mixer</td><td>VWR</td><td>945300</td><td>Speed 1,000-3,200 rpm</td></tr><tr><td>Homogenizer</td><td>IKA T10 Basic Ultra-Turbax</td><td>3420000</td><td></td></tr><tr><td>Mini-extruder</td><td>Avanti Polar Lipids</td><td>610020</td><td></td></tr><tr><td>Extruder filters</td><td>Whatman</td><td>610014</td><td>drain disc 10 mm&amp;nbsp;</td></tr><tr><td>Extruder polycarbonate membrane 400 nm</td><td>Whatman</td><td>61007</td><td>nuclepore polycarbonate&amp;nbsp;</td></tr><tr><td>Speed vacuum</td><td>Labconco</td><td>7970011</td><td>Centritrap DNA concentrator</td></tr><tr><td>PURExpress kit</td><td>New England Biolabs</td><td>NRM #E6800S</td><td></td></tr><tr><td>RNAse inhibitor (40,000 U/ml)</td><td>New England Biolabs</td><td>#M0307S</td><td></td></tr><tr><td>Proteinase K (20.2 mg/ml)</td><td>Fermentas</td><td>#EO0491</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Zeiss Observer Z1with a AxioCam MRm camera</td><td></td><td></td></tr><tr><td>RealTime</td><td>CFX96 Real time PCR Detection System (Biorad)</td><td></td><td></td></tr><tr><td>Silicon press to seal&amp;nbsp;&amp;nbsp;-Molecular Probe</td><td>Life Technologies</td><td>P18174</td><td>Resistant from&amp;nbsp; -25-30 &amp;deg;C</td></tr><tr><td>Siliconized glass circle cover slides</td><td>Hampton Research</td><td>HR3-231</td><td>&amp;nbsp;&amp;nbsp; Diameter= 22 mm</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr></tbody></table>

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.

Fluorescence microscopy reveals that fluorescence is only observed inside of the vesicles, because extravesicular material is enzymatically degraded (Figure 3). For the expression of mVenus, intravesicular fluorescence begins to be observed after 1.5 hr at 37 &#176;C and reaches maximum fluorescence intensity within 6 hr. The optimal temperature and incubation time can vary depending upon the specific constructs used. For example, different fluorescent proteins mature quite differently in a temperature dependent manner. In other words, the observation of protein production is not solely dependent upon protein synthesis and folding but also on chromophore formation. Overall protein synthesis can be increased by incorporating membrane protein pores to allow for an influx of depleted&#160;components necessary for transcription and translation27.
It is recommended to carry out an analogous transcription-translation reaction in the absence of vesicles to ensure that the exploited genetic construct is functional. This control reaction is more easily monitored by fluorescence spectroscopy rather than microscopy.&#160;Figure 4 shows an in vitro transcription-translation reaction of a construct encoding mVenus. Unencapsulated reactions give much higher total fluorescence intensities than similar intravesicular reactions. This is due to encapsulation efficiency and because the total intravesicular volume is much less than the extravesicular volume (i.e. a dilution effect).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig1.jpg" width="600px" /> 
Figure 1. The rotary evaporator and vacuum pump. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51304/51304fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig2.jpg" width="600px" /> 
Figure 2. The housing and parts of the extruder are shown separately. From left to right, syringe, retainer nut, teflon bearing, internal membrane supports with black O-rings facing one another, extruder outer casing, and second syringe. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51304/51304fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig3.jpg" width="600px" /> 
Figure 3. Fluorescence images of mVenus protein production in liposomes.&#160;A&#160;and&#160;C:&#160;Bright field images of multilamellar vesicles after 1.5 hr and 2.5 hr.&#160;B and&#160;D:&#160;The production of mVenus is visualized by fluorescence (colored green) after 1.5 and 2.5 hr, respectively. Scale bar is 20 &#956;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51304/51304fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51304/51304fig4.jpg" width="600px" /> 
Figure 4. In vitro control reaction of nonencapsulated in vitro transcription and translation of mVenus. Fluorescence intensity was measured every 5 min over 4 hr. The data were acquired with a Real-Time PCR instrument. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51304/51304fig4highres.jpg" target="_blank">Click here to view larger image</a>.

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

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 ...

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JoVE Journal

Bioengineering

15.0K Views.  University of Trento. 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. 

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

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

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 ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

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

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, ...

Biochemistry

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

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We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

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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.

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Immunology and Infection

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

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This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

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

Summary

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Chapters in this video

Sealable Femtoliter Chamber Arrays for Cell-free Biology

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

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

Cell-Free Protein Synthesis System for Building Synthetic Cells

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

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

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles