M. Y. acknowledges the funding from the Postgraduate Research &#38; Practice Innovation Program of Jiangsu Province, China (Grant No. KYCX22_2803). L.K. is thankful for the support of the Natural Science Research of Jiangsu Higher Education Institutions of China, China (Grant No. 17KJB180003), the Natural Science Foundation of Jiangsu Normal University, China (Grant No. 17XLR037), Priority Academic Program Development of Jiangsu Higher Education Institutions, China, and the Jiangsu Specially-Appointed Professor program, China.

1. Extract preparation
<ol>
	<li>Streak the E. coli BL21 (DE3) strain from a glycerol stock onto a Luria Bertani (LB) agar plate and incubate for at least 15 h at 37 &#176;C.</li>
	<li>Prepare an overnight preculture by inoculating a single colony from the freshly prepared LB plate into a 50 mL flask of Luria Bertani (LB) medium.</li>
	<li>Inoculate 5 mL of preculture into 500 mL of 2xYTPG medium in a 3 L baffled Erlenmeyer flask. Grow it at 37 &#176;C with vigorous shaking (between 220 rpm and 250 rpm) and harvest the cells when they reach the mid-log phase (OD600 ~ 3). Then, place the cell cultures in cold icy water for 10 min and centrifuge at 8,000 &#215; g for 15 min at 4 &#176;C. 
	NOTE: In this step, the glucose in 2xYTPG medium can be omitted for specific applications9,12.</li>
	<li>Resuspend the resulting cell pellets in 35 mL of prechilled S30 buffer A, followed by centrifugation at 8,000 &#215; g for 15 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Repeat step 1.4 twice. 
	NOTE: The cell pellets can be stored at -80 &#176;C if not used immediately.</li>
	<li>Resuspend the final pellets in S30 buffer B (e.g., use 1.1 mL of S30 buffer B per 1 g of cell pellet) and disrupt the cells by one pass through French Press at 17,000 psi.
	<ol>
		<li>For using the French Press, follow the user manual and transfer the cell suspension to the French press metal disruption chamber, ensuring that all components are assembled and the bottom valve of the disruption chamber is closed.</li>
		<li>Transfer the assembled disruption chamber to the hydraulic platform and secure the safety lock.</li>
		<li>Turn on the hydraulic pump and start the disruption.</li>
		<li>Control the outlet valve to enable the cell suspension to flow out of the outlet tube into a new 50 mL tube. Adjust the flow speed to ensure efficient disruption, ideally one drop at a time. Maintain the pressure above the lower limit of 15,000 psi.</li>
	</ol>
	</li>
	<li>Centrifuge the resulting lysate at 30,000 &#215; g for 30 min at 4 &#176;C and collect the supernatant. Repeat the centrifugation once and collect the supernatant. Add 0.3 volume of preincubation buffer into the collected supernatant and incubate with gentle shaking at 37 &#176;C for 80 min. 
	NOTE: The use of preincubation buffer could increase the final yield though it has been reported that an empty run-off (without preincubation buffer) could also improve the efficiency37,38, which depends on the corresponding source strain.</li>
	<li>Dialyze the resulting run off mixture for 2 h against 2 L of S30 buffer C, exchange the dialysis buffer once with 2 L of fresh S30 buffer C, and dialyze overnight at 4 &#176;C39. 
	NOTE: The second step of overnight dialysis can be shortened to approximately 3 h.</li>
	<li>Collect the dialyzed sample and centrifuge at 30,000 &#215; g for 30 min at 4 &#176;C. Collect the supernatant and aliquot into appropriate volumes and flash freeze immediately in liquid nitrogen. 
	NOTE: The frozen sample can be stored at -80 &#176;C at least for 6 months to 1 year without loss of efficiency.</li>
</ol>
2. T7 RNA polymerase
<ol>
	<li>Transform pAR1219 plasmid into BL21(DE3) star E. coli competent cells40.</li>
	<li>Inoculate 10 mL of an overnight culture into 1 L of LB medium (containing 100 &#181;g/mL ampicillin). Grow the cells at 37 &#176;C until OD600 reaches 0.6-0.8.</li>
	<li>Start the induction by adding a final concentration of 1 mM IPTG. Induce the cells for an additional 5 h and harvest by centrifugation at 8,000 &#215; g for 15 min at 4 &#176;C. Store the cell pellets at -80 &#176;C for up to several weeks.</li>
	<li>Resuspend the cell pellets in 30 mL of T7 buffer A and disrupt the cells by one pass through the French Press at 15,000 psi. Remove the cell debris by centrifugation at 20,000 &#215; g for 30 min at 4 &#176;C.</li>
	<li>Add streptomycin sulfate drop by drop to the supernatant from the previous step, reaching a final concentration of 4% (w/v). After a short incubation on ice, centrifuge at 20,000 &#215; g for 30 min at 4 &#176;C.</li>
	<li>Filter the supernatant through a 0.45 &#181;m filter membrane and load the filtered sample onto a strong anion exchange column (column volume of 40 mL), which was preequilibrated with 10 column volumes of T7 buffer B, via an automated liquid chromatograph system.</li>
	<li>Wash the loaded column with 50 column volumes of T7 buffer B and elute the sample with 10 column volumes of low salt (50 mM NaCl) and high salt (500 mM) T7 buffer C mixtures, establishing a linear concentration gradient of NaCl from 50 to 500 mM at a flow rate of ~3 mL/min41.</li>
	<li>Collect the peak fractions and analyze by SDS-PAGE. 
	NOTE: T7 polymerase exhibits a predominate band at ~100 kDa, while significant amounts of impurities still appear on the SDS-PAGE gel.</li>
	<li>Pool the fractions containing T7 polymerase and dialyze against 2 L of T7 buffer C overnight.</li>
	<li>Add glycerol to reach a final concentration of 10% and concentrate the resulting mixture to a final concentration of 3-4 mg/mL by ultrafiltration42. 
	NOTE: If precipitation appears during the concentration process, stop immediately and remove the precipitates by centrifugation.</li>
	<li>Adjust the glycerol concentration to 50%. Aliquot and flash freeze the sample with liquid nitrogen. Store at &#8722;80 &#176;C for a longer period. 
	NOTE: A small aliquot could also be stored at -20 &#176;C for at least 1 month without loss of efficiency.</li>
</ol>
3. Buffer preparation
<ol>
	<li>Prepare buffers as indicated in Table 1 a day before usage.</li>
</ol>
4. Template design and preparation
<ol>
	<li>Clone the gene of interest into a T7 promoter-based vector or generate a linear PCR product containing the gene of interest. 
	NOTE: See the discussion section for design principles.</li>
	<li>Prepare the plasmid from an overnight culture using a plasmid extraction kit. 
	NOTE: We recommend selecting a plasmid kit that includes an isopropanol precipitation step followed by a washing procedure with 70% ethanol.</li>
	<li>Dissolve the dried DNA in a small volume of ultrapure water to a concentration between 200 &#181;g/mL and 500 &#181;g/mL, as determined by a micro volume spectrophotometer.</li>
	<li>Optional: Directly express proteins from linear PCR products. 
	NOTE: In this case, a PCR purification step is needed.</li>
</ol>
5. Mg2+ and K+ optimization
<ol>
	<li>Prepare a master mix for Mg2+ concentration screening as indicated in Table 2, or for K+ concentration screening as indicated in Table 3.</li>
	<li>Transfer the master mix into individual microfuge tubes or a V-shaped 96-well plate.</li>
	<li>Pipette the exact volume of Mg2+ or K+ stock solutions into individual microfuge tubes or the V-shaped 96-well plate and complete the CFPS reactions with ultrapure water. Incubate the reactions for at least 2 h. 
	NOTE: If using a V-shaped 96-well plate, seal the plate with a plastic cover to prevent evaporation.</li>
	<li>Take out 2 &#181;L of the reaction mixture and transfer into a black 96-well plate for fluorescence measurements by a plate reader. 
	NOTE: We use a fluorescent plate reader with the following setup: excitation/emission: 485/528, Gain: 50, Read height: 7.00 mm. However, one would need to tune their plate reader to generate a specific calibration curve using purified fluorescent proteins.</li>
</ol>
6. Encapsulation
<ol>
	<li>Droplet
	<ol>
		<li>Prepare a surfactant-containing fluorinated oil (2% PFPE-PEG in HFE7500) or a lipid-mineral oil solution by dissolving lipid in mineral oil (refer to step 6.2.1)</li>
		<li>Prepare a total volume of 100 &#181;L of CFPS reaction by combining the corresponding reagents 2-18 from Table 4. Add CFPS reaction to 500 &#181;L of the previously prepared oil in a 1.5 mL tube and then, rub the tube vigorously on the tube rack 50x to form fine (water-in-oil) droplets. Incubate the tube at 30 &#176;C to perform the reaction. 
		NOTE: Simple microfluidics chips could also be used to generate homogenous droplets as well43.</li>
	</ol>
	</li>
	<li>Giant unilamellar vesicles (GUVs)
	<ol>
		<li>Preparing the lipid-mineral oil solution
		<ol>
			<li>Add 57 &#181;L of chloroform into a 4 mL glass vial and then add 18 &#181;L of 25 mg/mL 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC) lipids to form the chloroform lipid solution, achieving a final concentration of 8 mM (use other lipids with similar final concentrations). 
			NOTE: This mixing step was to ensure homogeneous mixing of different lipids.</li>
			<li>Evaporate the chloroform under an argon flow for 15 min, and then, further evaporate under vacuum for 1 h.</li>
			<li>Dissolve the resulting dry lipids in 1,500 &#181;L of mineral oil, reaching a final concentration of 400 &#181;M. Incubate the mixture overnight at room temperature.</li>
		</ol>
		</li>
		<li>Forming an interfacial lipid layer
		<ol>
			<li>Add 500 &#181;L of the outer solution (see Table 5) to a 1.5 mL tube, and slowly layer 250 &#181;L of lipid-mineral oil solution on top of the outer solution. Incubate at room temperature for 30 min to form a stable interfacial lipid layer.</li>
		</ol>
		</li>
		<li>Preparation of the inner solution
		<ol>
			<li>Mix all the reagents in Table 6 to form the preinner solution and keep it on ice until use. Complete the inner solution by adding T7RNAP, S30 extract, and DNA template into the preinner solution as reagents 16-18 listed in Table 4. 
			NOTE: Increasing encapsulated sucrose concentration above 250 mM might inhibit cell-free translation44.</li>
		</ol>
		</li>
		<li>Formation of GUVs
		<ol>
			<li>Add 50 &#181;L of inner solution to a 1.5 mL new tube containing 500 &#181;L of lipid-mineral oil, pipette up and down rapidly, and vortex vigorously.</li>
			<li>Leave the tube on ice for 10 min and slowly add 20 &#181;L of the emulsion mixture to the top of the oil phase in the 1.5 mL tube from step 6.2.2.</li>
			<li>Centrifuge at 800 &#215; g (use a centrifugation speed ranging from 100 &#215; g to 1000 &#215; g) for 10 min at 4 &#176;C and observe the formation of GUVs at the bottom of the tube. 
			NOTE: A higher specific centrifugation speed could be used; however, one would consider that the centrifugation speed could influence the size of formed GUVs45.</li>
			<li>Remove the upper oil phase.</li>
			<li>Aspirate 30 &#181;L of GUVs at the bottom of the tube carefully. Incubate the collected GUVs at 30 &#176;C. 
			NOTE: The optimal incubation temperature varies for different proteins.</li>
			<li>Use Confocal Laser Scanning Microscopy (LSM) to monitor the expression of fluorescent proteins.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Supported lipid bilayer (SLB)
	<ol>
		<li>Preparation of SLB chambers
		<ol>
			<li>Piranha-clean 24 x 24 mm #1.5 coverslips by adding seven drops of sulfuric acid and two drops of 50% hydrogen peroxide to the center of each cover slide. Incubate the reactants on the coverslips for at least 45 min; rinse thoroughly with ultrapure water.</li>
			<li>Assemble the reconstitution chamber by attaching a cut 0.5 mL microfuge tube onto the cleaned coverslips using optical glue cured under an ultraviolet lamp (365 nm) for 10 min to form a reaction chamber.</li>
		</ol>
		</li>
		<li>SLB formation
		<ol>
			<li>Dissolve 80 mol% 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 19.95 mol% 1,2-dioleoyl-sn-glycerol-3-phospho-L-serine (DOPS), and 0.05 mol% Atto488-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine = DOPE), mix in chloroform, dry under mild nitrogen flow and vacuum for 1 h to remove the solvent completely.</li>
			<li>Rehydrate the dried lipid film in SLB buffer A, resulting in a final lipid concentration of 4 mg/mL.</li>
			<li>Vortex and sonicate the resulting samples until they become clear.</li>
			<li>Dilute 4 mg/mL of SUVs with 130 &#181;L of SLB buffer A, transfer 75 &#181;L of the suspension to the preformed reaction chambers, and incubate it on a heat block at 37 &#176;C for 1 min. Add 150 &#181;L of SLB buffer A to the reaction chamber for further incubation for 2 min.</li>
			<li>Wash the chamber with 2 mL of SLB buffer B (SLB buffer A without MgCl2) prior to a buffer exchange to the S30 buffer C with 0.4% (w/v) BSA for CFPS reactions, leaving 100 &#181;L of buffer inside the chamber to prevent the formed SLB from drying out.</li>
			<li>Remove the residual S30 buffer C and add the CFPS reaction mixture into the SLB chamber carefully. Incubate the chamber at 30 &#176;C and monitor the expression under a confocal laser scanning microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This manuscript outlines a modified Cell-Free Protein Synthesis (CFPS) system designed for use in various micro-compartments across synthetic cell platforms, including water-in-oil droplets, GUVs, and SLBs. We utilized the standard E. coli recombinant protein expression host strain, BL21(DE3), as the source extract for constructing protein-centric synthetic cell systems. This approach yielded approximately 0.5 mg/mL of protein across different compartments. While other customized extract source strains could be ...

The synthesis of synthetic or artificial cells has emerged as a highly prominent field of interdisciplinary research, attracting substantial interest from scientists across the domains of synthetic biology, chemistry, and biophysics. These scientists are united by the common goal of constructing a minimal living cell1,2,3. The rapid growth of this field has been in step with significant advancements in critical technologies, such as recombinant DNA manipulation4, biomimetic materials5, and microfabrication techniques for compartmentalization6, including the Cell-Free Protein Synthesis (CFPS) method7. CFPS systems encompass the essential cellular machinery for transcription and translation, providing the foundational framework for the development and integration of multifunctional artificial cells.
Although CFPS techniques are frequently used in the assembly of synthetic cells, developing a robust and tailored CFPS system for the assembly of various synthetic cell systems remains a complex challenge. Currently, numerous CFPS systems are available, derived from both prokaryotes and eukaryotes model organisms8, each specialized for particular applications in synthetic cell synthesis. Beyond their central roles in transcription and translation, CFPS systems vary in their main components and associated preparation procedures. These variations, which include differences in cell extracts, RNA polymerases, template preparation methods, and buffer compositions, are largely due to the distinct development trajectories pursued by research groups that have intensively optimized their systems for maximal protein yield.
Among the various components of the CFPS system, the cell extract is a critical enzymatic pool for transcription and translation, and thus a key determinant of CFPS performance9. Escherichia coli (E. coli)-based CFPS is the most commonly utilized system due to its status as the best-understood prokaryotic organism. Furthermore, a fully reconstituted CFPS system comprising individually purified proteins and ribosomes, known as PURE10, has been developed by Ueda&#39;s research group, which is particularly suited for applications requiring a clear background. Today, even E. coli-based CFPS systems have diversified, especially in terms of the source strains for the extrac11 and methods of preparation12,13, RNA polymerase14,15, energy sources16,17, and buffer systems18,19. The most frequently used strains include K12 and B strain derivatives, such as A1920, JM10921, BL21 (DE3)22, and Rossetta223, alongside their genetically modified counterparts.
Initially, E. coli strains with reduced RNase and protease activities were chosen to enhance mRNA stability and the stability of newly synthesized recombinant proteins, leading to increased final protein yields24. Subsequently, E. coli extracts were engineered to facilitate specific post-translational modifications, including glycosylation25, phosphorylation26, and lipidation27, were developed to achieve the above posttranslational modifications. Additionally, an array of additives such as molecular chaperons28 and chemical stabilizers have been incorporated to aid the folding of target proteins, contributing to the diversification of CFPS systems. The bacteriophage T7 RNA polymerase, known for its high processivity, is predominantly employed for transcription, although other polymerases such as SP629 have also been utilized. E. coli endogenous RNA polymerase has been adapted for the prototyping of genetic circuits leveraging sigma factors30. Lastly, a variety of energy precursors31,32,33 and different salts and buffer components19,34,35 have been systematically optimized to enhance productivity.
Besides the CFPS system itself, the encapsulation methods as well as compartmentalization materials are also vital for the successful synthetic cell assembly. Various systems that have been developed to successfully encapsulate the CFPS reaction include surfactant-stabilized water/oil droplets, lipid/polymer, and their hybrid unilamellar vesicles (with diameters ranging from 50 nm to several &#956;m), as well as planar-supported lipid bilayers. However, due to the complexed molecule content of the CFPS system, the success rate of encapsulation depends on specific cases, particularly for the formation of vesicles. To improve the success rate and efficiency of encapsulation of CFPS, various microfluid chips have been developed to facilitate the formation of both droplets and vesicles36. Nevertheless, additional chips and devices will need to be established.
This protocol delineates an E. coli CFPS system utilizing the BL21(DE3) strain, which is a commonly employed host for recombinant protein production. The protocol encompasses a detailed account of the cell extract preparation, template preparation, and standard expression optimization using a fluorescent reporter protein. Moreover, we present exemplary outcomes achieved by encapsulating the CFPS system within diverse micro-compartments, including monolayer droplets, double emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we expound upon the pivotal procedural elements and the requisite conditions indispensable for the successful establishment of these CFPS systems within distinct environmental contexts.

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine(DOPC)</td><td>Avanti</td><td>850375P</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-L-serine (sodium salt)(DOPS)</td><td>Avanti</td><td>840035P</td><td></td></tr><tr><td>1,4 dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>1.11474</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td><td>Avanti</td><td>850457P</td><td></td></tr><tr><td>3,5-cyclic AMP (cAMP)</td><td>Sigma-Aldrich</td><td>A9501</td><td></td></tr><tr><td>50 mL tubes</td><td>Eppendorf</td><td>Eppendorf Tubes BioBased</td><td></td></tr><tr><td>50% hydrogen peroxide</td><td>Sigma-Aldrich</td><td>516813</td><td></td></tr><tr><td>Acetate</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Agar powder</td><td>Sigma-Aldrich</td><td>05040</td><td></td></tr><tr><td>Alanin</td><td>Sigma-Aldrich</td><td>A4349</td><td></td></tr><tr><td>Amicon Stirred Cells</td><td>MerckMillipore</td><td>UFSC05001</td><td></td></tr><tr><td>Ammonium acetate (NH4OAc)</td><td>Sigma-Aldrich</td><td>A7262</td><td></td></tr><tr><td>Arginin</td><td>Sigma-Aldrich</td><td>A4474</td><td></td></tr><tr><td>Asparagin</td><td>Sigma-Aldrich</td><td>A0884</td><td></td></tr><tr><td>Aspartat</td><td>Sigma-Aldrich</td><td>A5474</td><td></td></tr><tr><td>ATP</td><td>Roche</td><td>11140965001</td><td></td></tr><tr><td>Atto 488 DOPE</td><td>Sigma-Aldrich</td><td>67335</td><td></td></tr><tr><td>Atto 647N DOPE</td><td>Sigma-Aldrich</td><td>42247</td><td></td></tr><tr><td>Baffled Erlenmeyer flask</td><td>Shuniu</td><td>250 mL, 1000mL</td><td></td></tr><tr><td>Bovine Serum Albumin(BSA)</td><td>Roche</td><td>10711454001</td><td></td></tr><tr><td>Centrifugetube</td><td>Eppendorf</td><td>Eppendorf Tubes 3810X</td><td></td></tr><tr><td>Centrifugetube rack</td><td>Eppendorf</td><td>0030119819</td><td></td></tr><tr><td>Chemiluminescence and epifluorescence imaging system</td><td>Uvitec</td><td>Alliance Q9 Advanced</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306</td><td></td></tr><tr><td>Confocal Laser Scanning Microscopy (LSM)</td><td>ZEISS</td><td>LSM 780</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Thermo Fisher Scientific</td><td>C10283</td><td></td></tr><tr><td>Coverslip</td><td>Thermo Scientific</td><td>Menzel BB02400500A113MNZ0</td><td></td></tr><tr><td>creatine kinase (CK)</td><td>Roche</td><td>10127566001</td><td></td></tr><tr><td>Creatine phosphate (CP)</td><td>Sigma-Aldrich</td><td>10621714001</td><td></td></tr><tr><td>Culture dish</td><td>Huanqiu</td><td>90 mm</td><td></td></tr><tr><td>Cystein</td><td>Sigma-Aldrich</td><td>C5360</td><td></td></tr><tr><td>Cytidine 5&#039;-triphosphate disodium salt (CTP)</td><td>aladdin</td><td>C101487</td><td></td></tr><tr><td>Dialysis membrane</td><td>Spectrum</td><td>Standard RC Tubing MWCO: 12-14 kD</td><td></td></tr><tr><td>E.Z.N.A. Cycle Pure Kit</td><td>Omega Bio-Tek</td><td>D6492-01</td><td></td></tr><tr><td>Electro-Heating Standing-Temperature Cultivator</td><td>Yiheng instrument</td><td>DHP-9602</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid(EDTA)</td><td>Biosharp</td><td>1100027</td><td></td></tr><tr><td>Fluorescent plate reader</td><td>BioTek</td><td>Synergy 2</td><td></td></tr><tr><td>Fluorinated oil</td><td>Suzhou CChip scientific instrument</td><td>2%HFE7500</td><td></td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich</td><td>47612</td><td></td></tr><tr><td>French Press</td><td>G.Heinemann</td><td>HTU-DIGI-Press</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glutamat</td><td>Sigma-Aldrich</td><td>G5667</td><td></td></tr><tr><td>Glutamin</td><td>Sigma-Aldrich</td><td>G5792</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Glycin</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate(GTP)</td><td>Roche</td><td>10106399001</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HiPrep Q FF 16/10</td><td>Cytiva</td><td>28936543</td><td></td></tr><tr><td>Histidin</td><td>Sigma-Aldrich</td><td>H6034</td><td></td></tr><tr><td>Isoleucin</td><td>Sigma-Aldrich</td><td>I5281</td><td></td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P8281</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Leucin</td><td>Sigma-Aldrich</td><td>L6914</td><td></td></tr><tr><td>Lysin</td><td>Sigma-Aldrich</td><td>L5501</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate (Mg(OAc)&lt;sub&gt;2&lt;/sub&gt; )</td><td>Sigma-Aldrich</td><td>M5661</td><td></td></tr><tr><td>Magnesium chloride(MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Methionin</td><td>Sigma-Aldrich</td><td>M8439</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424 R</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M5904</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell Systems</td><td>Bio-Rad</td><td>1645050</td><td></td></tr><tr><td>Multipurpose Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>NaN3</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Nucleic Acid &amp;amp; Protein UV-Assay Measurements</td><td>IMPLEN</td><td>NanoPhotometer N60</td><td></td></tr><tr><td>NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA</td><td>MACHEREY-NAGEL</td><td>740414.5</td><td></td></tr><tr><td>Nunc-Immuno MicroWell 96 well polystyrene plates</td><td>Sigma-Aldrich</td><td>P8616</td><td></td></tr><tr><td>PCR Thermal Cycler</td><td>Eppendorf</td><td>Mastercycler nexus</td><td></td></tr><tr><td>Peptone</td><td>Sigma-Aldrich</td><td>83059</td><td></td></tr><tr><td>Phenylalanin</td><td>Sigma-Aldrich</td><td>P8740</td><td></td></tr><tr><td>Phosphoenolpyruvat (PEP)</td><td>GLPBIO</td><td>GC44635</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>PMSF-RO</td><td></td></tr><tr><td>Polyethylene glycol 8000 (PEG 8000)</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>Potassium Acetate(KOAc)</td><td>Sigma-Aldrich</td><td>P5708</td><td></td></tr><tr><td>Potassium chloride(KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Potassium glutamate (K-glutamate)</td><td>Sigma-Aldrich</td><td>G1501</td><td></td></tr><tr><td>Potassium hydroxide(KOH)</td><td>Sigma-Aldrich</td><td>221473</td><td></td></tr><tr><td>Prolin</td><td>Sigma-Aldrich</td><td>P8865</td><td></td></tr><tr><td>Pyruvate kinase (PK)</td><td>Sigma-Aldrich</td><td>P9136</td><td></td></tr><tr><td>Serin</td><td>Sigma-Aldrich</td><td>S4311</td><td></td></tr><tr><td>Shaker</td><td>Zhichushakers</td><td>ZQZY-AF8</td><td></td></tr><tr><td>Sodium chloride(NaCl)</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sodium hydroxide(NaOH)</td><td>Sigma-Aldrich</td><td>S5881</td><td></td></tr><tr><td>Sucrose</td><td>aladdin</td><td>S112226</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td></td></tr><tr><td>Syringe Filters</td><td>Jinteng</td><td>0.45 &amp;mu;m</td><td></td></tr><tr><td>Test tube</td><td>Shuniu</td><td>20 mL</td><td></td></tr><tr><td>TGX FastCast Acrylamide Kit, 12%</td><td>Bio-Rad</td><td>#1610175</td><td></td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>ThermoMixer C</td><td></td></tr><tr><td>Threonin</td><td>Sigma-Aldrich</td><td>T8441</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>V900483</td><td></td></tr><tr><td>tRNA</td><td>Roche</td><td>10109550001</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293</td><td></td></tr><tr><td>Tryptophan</td><td>Sigma-Aldrich</td><td>T8941</td><td></td></tr><tr><td>Tyrosin</td><td>Sigma-Aldrich</td><td>T8566</td><td></td></tr><tr><td>UTP Trisodium salt (UTP)</td><td>aladdin</td><td>U100365</td><td></td></tr><tr><td>Vacuum Pump with Circulated Water System</td><td>Zhengzhou Greatwall Scientific Industrial and Trade Co.Ltd</td><td>SHB-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66626/66626eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Valin</td><td>Sigma-Aldrich</td><td>V4638</td><td></td></tr><tr><td>Vortex Mixers</td><td>Kylin-Bell</td><td>Vortex QL-861</td><td></td></tr><tr><td>Water purification system</td><td>MerckMillipore</td><td>Direct ultrapure water (Type 1)</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>70161</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>444203</td><td></td></tr></tbody></table>

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.

For each new batch of cell extract and T7 RNA polymerase, it is recommended to perform a basic screening of both Mg2+ and K+ concentrations to ensure the optimal performance of the CFPS system. The fluorescence of superfolder GFP can serve as an indicator of the overall yield of the CFPS system under varying conditions, as illustrated in Figure 1A,B. Additionally, a parallel yield comparison of the CFPS system across different compartments is shown in <...

Read this Scientific Article Protocol about Cell-free Protein Synthesis System for Building Synthetic Cells at JoVE.com

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

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 ...

cell-free-protein-synthesis-system-for-building-synthetic-cells

Research

JoVE Journal

Biochemistry

2.4K Views.  Jiangsu Normal University. Al ons onderzoek richt zich op celvrije synthetische biologie. Dit omvat bottom-up assemblage van synthetische cellen, functionele en gestructureerde analyse van membraaneiwitten, eiwitengineering terwijl het incorporeren van niet-conische aminozuren en celvrije metabole engineering omvat. Momenteel naast celvrije eiwitsynthese, micro for edicts apparaat door microfabricage door middel van zachte lithografie, ontwikkeling van biocompatibele materialen en de a...

Video: Auteur in de schijnwerpers: CFPS-systemen optimaliseren voor de bouw van synthetische cellen

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

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

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

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

Bioengineering

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Chemistry

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

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 ...

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 ...

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

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...

Cell-Free Protein Synthesis System for Building Synthetic Cells

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