Cell-free Protein Synthesis System for Building Synthetic Cells

Abstract

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.

Introduction

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 cell^1,2,3. 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'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 preparation^12,13, RNA polymerase^14,15, energy sources^16,17, and buffer systems^18,19. 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 precursors^31,32,33 and different salts and buffer components^19,34,35 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 μm), 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.

Protocol

1. Extract preparation

1. 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 °C.
2. 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.
3. Inoculate 5 mL of preculture into 500 mL of 2xYTPG medium in a 3 L baffled Erlenmeyer flask. Grow it at 37 °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 × g for 15 min at 4 °C.
   NOTE: In this step, the glucose in 2xYTPG medium can be omitted for specific applications^9,12.
4. Resuspend the resulting cell pellets in 35 mL of prechilled S30 buffer A, followed by centrifugation at 8,000 × g for 15 min at 4 °C. Discard the supernatant.
5. Repeat step 1.4 twice.
   NOTE: The cell pellets can be stored at -80 °C if not used immediately.
6. 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.
   1. 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.
   2. Transfer the assembled disruption chamber to the hydraulic platform and secure the safety lock.
   3. Turn on the hydraulic pump and start the disruption.
   4. 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.
7. Centrifuge the resulting lysate at 30,000 × g for 30 min at 4 °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 °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 efficiency^37,38, which depends on the corresponding source strain.
8. 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 °C³⁹.
   NOTE: The second step of overnight dialysis can be shortened to approximately 3 h.
9. Collect the dialyzed sample and centrifuge at 30,000 × g for 30 min at 4 °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 °C at least for 6 months to 1 year without loss of efficiency.

2. T7 RNA polymerase

1. Transform pAR1219 plasmid into BL21(DE3) star E. coli competent cells⁴⁰.
2. Inoculate 10 mL of an overnight culture into 1 L of LB medium (containing 100 µg/mL ampicillin). Grow the cells at 37 °C until OD₆₀₀ reaches 0.6-0.8.
3. 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 × g for 15 min at 4 °C. Store the cell pellets at -80 °C for up to several weeks.
4. 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 × g for 30 min at 4 °C.
5. 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 × g for 30 min at 4 °C.
6. Filter the supernatant through a 0.45 µm 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.
7. 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.
8. 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.
9. Pool the fractions containing T7 polymerase and dialyze against 2 L of T7 buffer C overnight.
10. 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.
11. Adjust the glycerol concentration to 50%. Aliquot and flash freeze the sample with liquid nitrogen. Store at −80 °C for a longer period.
    NOTE: A small aliquot could also be stored at -20 °C for at least 1 month without loss of efficiency.

3. Buffer preparation

1. Prepare buffers as indicated in Table 1 a day before usage.

4. Template design and preparation

1. 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.
2. 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.
3. Dissolve the dried DNA in a small volume of ultrapure water to a concentration between 200 µg/mL and 500 µg/mL, as determined by a micro volume spectrophotometer.
4. Optional: Directly express proteins from linear PCR products.
   NOTE: In this case, a PCR purification step is needed.

5. Mg²⁺ and K⁺ optimization

1. Prepare a master mix for Mg²⁺ concentration screening as indicated in Table 2, or for K⁺ concentration screening as indicated in Table 3.
2. Transfer the master mix into individual microfuge tubes or a V-shaped 96-well plate.
3. 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.
4. Take out 2 µL 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.

6. Encapsulation

1. Droplet
   1. 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)
   2. Prepare a total volume of 100 µL of CFPS reaction by combining the corresponding reagents 2-18 from Table 4. Add CFPS reaction to 500 µL 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 °C to perform the reaction.
      NOTE: Simple microfluidics chips could also be used to generate homogenous droplets as well⁴³.
2. Giant unilamellar vesicles (GUVs)
   1. Preparing the lipid-mineral oil solution
      1. Add 57 µL of chloroform into a 4 mL glass vial and then add 18 µL 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.
      2. Evaporate the chloroform under an argon flow for 15 min, and then, further evaporate under vacuum for 1 h.
      3. Dissolve the resulting dry lipids in 1,500 µL of mineral oil, reaching a final concentration of 400 µM. Incubate the mixture overnight at room temperature.
   2. Forming an interfacial lipid layer
      1. Add 500 µL of the outer solution (see Table 5) to a 1.5 mL tube, and slowly layer 250 µL 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.
   3. Preparation of the inner solution
      1. 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.
   4. Formation of GUVs
      1. Add 50 µL of inner solution to a 1.5 mL new tube containing 500 µL of lipid-mineral oil, pipette up and down rapidly, and vortex vigorously.
      2. Leave the tube on ice for 10 min and slowly add 20 µL of the emulsion mixture to the top of the oil phase in the 1.5 mL tube from step 6.2.2.
      3. Centrifuge at 800 × g (use a centrifugation speed ranging from 100 × g to 1000 × g) for 10 min at 4 °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.
      4. Remove the upper oil phase.
      5. Aspirate 30 µL of GUVs at the bottom of the tube carefully. Incubate the collected GUVs at 30 °C.
         NOTE: The optimal incubation temperature varies for different proteins.
      6. Use Confocal Laser Scanning Microscopy (LSM) to monitor the expression of fluorescent proteins.
3. Supported lipid bilayer (SLB)
   1. Preparation of SLB chambers
      1. 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.
      2. 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.
   2. SLB formation
      1. 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.
      2. Rehydrate the dried lipid film in SLB buffer A, resulting in a final lipid concentration of 4 mg/mL.
      3. Vortex and sonicate the resulting samples until they become clear.
      4. Dilute 4 mg/mL of SUVs with 130 µL of SLB buffer A, transfer 75 µL of the suspension to the preformed reaction chambers, and incubate it on a heat block at 37 °C for 1 min. Add 150 µL of SLB buffer A to the reaction chamber for further incubation for 2 min.
      5. 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 µL of buffer inside the chamber to prevent the formed SLB from drying out.
      6. Remove the residual S30 buffer C and add the CFPS reaction mixture into the SLB chamber carefully. Incubate the chamber at 30 °C and monitor the expression under a confocal laser scanning microscope.

Representative Results

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 Figure 1C, demonstrating consistent performance in encapsulated CFPS systems, albeit with a slight decrease in GUVs, potentially due to the influence of sucrose⁴⁶. Furthermore, CFPS reactions were successfully encapsulated in droplets (Figure 2), GUVs (Figure 3), and atop SLBs (Figure 4).

Figure 2A highlights the formation of emulsion droplets, evident when the oil solution became turbulent. The expression of green/red fluorescent proteins (GFP and mCherry) was captured using a confocal laser scanning microscopy. Figure 3 showcased the success of the encapsulation of CFPS reaction within GUVs through the simple emulsion transfer process. The red channel indicated the labeled lipids; the green channel showed the expression of superfolder GFP; and the yellow channel represented an inert fluorescent dye. Figure 4A shows the SLB chamber where the CFPS reaction could be deposited. Within this chamber, an SLB was formed on top of the glass coverslip, with the CFPS reaction mixture placed atop the preformed SLB, enabling imaging via a confocal laser scanning microscopy. As indicated in Figure 4B, SLB was visualized by labeled lipids, shown in green, while the expressed small GTPase Cdc42 fused with mCherry was shown in magenta. The in-situ expression of mCherry-Cdc42 was observable, along with the reversible membrane targeting process, under the microscope. However, the CFPS system utilized was supplemented with prenylation machinery, as detailed in a recent publication⁴⁷.

Figure 1: Optimization and comparison of the CFPS system. Screening of (A) Mg²⁺ concentration and (B) K⁺ concentrations for the CFPS system in bulk. (C) A bar chart showing protein expression yields in different compartments. The fluorescence of superfolder GFP was used to determine the yield of the CFPS system, measured by a plate reader or a confocal laser scanning microscope. Abbreviations: CFPS = cell-free protein synthesis; GFP = green fluorescent protein; sfGFP = superfolder GFP; GUV = giant unilamellar vesicle; SLB = supported lipid bilayer. Please click here to view a larger version of this figure.

Figure 2: Cell-free protein synthesis in droplets. (A) A photo of encapsulated CFPS reactions in surfactant-stabilized water-in-oil droplets. (B) Brightfield and (C,D) fluorescence images of CFPS of (C) mCherry and (D) superfolder GFP encapsulated in droplets visualized by confocal laser scanning microscopy. Superfolder GFP is indicated in green; mCherry is indicated in magenta. Scale bars = 20 µm. Abbreviations: CFPS = cell-free protein synthesis; GFP = green fluorescent protein. Please click here to view a larger version of this figure.

Figure 3: Cell-free protein synthesis in GUVs. (A) A photo of encapsulated CFPS reactions in GUVs. (B-D) Fluorescence image of encapsulated CFPS within POPC GUVs, expressing superfolder GFP. (B) The lipid bilayer was labeled with 0.05% labeled Atto647N-DOPE lipids, shown in red, (C) an inert dye shown in yellow, and (D) superfolder GFP was indicated in green. Scale bars = 20 µm. Abbreviations: CFPS = cell-free protein synthesis; GFP = green fluorescent protein; GUV = giant unilamellar vesicle; POPC = 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine; DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. Please click here to view a larger version of this figure.

Figure 4: Cell-free protein synthesis of small GTPase protein-Cdc42 on top of SLBs. (A) Schematic illustration of the reconstitution of Cdc42's membrane targeting and RhoGDI-dependent membrane extraction on SLBs, as visualized by confocal laser scanning microscopy. The prenylation machinery was implemented via a prenlytransferase-enriched extract, which is detailed described elsewhere.⁴⁷. (B) An orthogonal view of the pre- and postprenylation of the reaction mixture on top of SLB. (C,D) Time series of mCherry-Cdc42 intensity on the membrane (pink) and in solution (black) during (C) membrane targeting and (D) membrane extraction. Scale bars = 10 µm. (A-D) were adapted from Kai et al.⁴⁷. Please click here to view a larger version of this figure.

Table 1: List of buffers. NOTE: Add β-mercaptoethanol and DTT just before use. Please click here to download this Table.

Table 2: Pipetting scheme for Mg²⁺ concentration screening. Please click here to download this Table.

Table 3: Pipetting scheme for K⁺ concentration screening. Please click here to download this Table.

Table 4: Inner-solution composition. Please click here to download this Table.

Table 5: Outer solution composition. Please click here to download this Table.

Table 6: Preinner solution composition. Please click here to download this Table.

Discussion

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 used, these systems have been primarily optimized for a specific type of protein or metabolic activity, which may be suboptimal for general protein expression. Recent advancements in quantitative mass spectrometry analysis have facilitated pilot studies on the proteomic analysis of cell lysate^48,49, particularly under various preparation conditions.

Despite gaining insights from these studies, establishing a quantitative correlation between these identified protein factors and the overall performance of CFPS systems remains challenging. This gap left unanswered questions about how to quantitatively assess the performance of a given batch of extract. Additionally, there has been no systematic and parallel comparison of different source strains reported. In this study, we utilized 2xYPTG medium to boost biomass production, a strategy recommended to transition to 2xYPT when utilizing endogenous RNA polymerase for transcription. Despite suggestions that T7 polymerase could be integrated into the extract preparation procedure⁴⁵, we opted for a separate preparation protocol for S30 extract and T7 RNA polymerase to reduce high batch-to-batch variations.

The production of target protein is intrinsically linked to the central transcription/translation process. Proper template design and purity are crucial for successful cell-free expression. In addition to the plasmid template, linear double-stranded DNA generated by PCR can also be employed, offering suitability for screening and high-throughput applications. Unlike conventional template design for in vivo expression in E. coli, template design in the E. coli cell-free system is simplified without the need for expression induction considerations. The only essential element of a cell-free expression template is a T7 regulatory sequence. However, in some instances, the 5' sequence of the target gene could influence the translation initiation, depending on the secondary structure formed by the corresponding transcribed mRNA. If the ribosome-binding site is located within a stable stem loop of the transcribed mRNA, translation initiation could be significantly hindered, leading to poor overall expression. Similarly, consecutive rare codons can hinder expression and should be optimized, similar to in vivo studies. This can be achieved through open-source servers dedicated to codon optimization⁵⁰ and mRNA secondary structure prediction⁵¹.

Regarding the buffer system, we utilized an acetate buffer supplemented with creatine phosphate and creatine kinase as the sources of energy regeneration in our experimental setup. This setup yielded a moderate expression level and a relatively fast kinetic, attributable to the single-step energy regeneration reaction catalyzed by creatine kinase using creatine phosphate. While some researchers have shifted to a glutamate buffer system, citing its closer resemblance to the cellular environment⁵², we observed no significant differences in protein expression yields with our current protocols. However, recent proteomic analysis indicated that different buffer systems in combination with extract processing methods could influence the final expression yield¹⁹. Concerning energy precursors, diverse substrates have been investigated by numerous research groups to achieve cost-effectiveness and heightened expression yields^53,54. In the context of the presented CFPS system, creatine phosphate and creatine kinase were deliberately chosen. This selection was motivated by the efficiency of energy regeneration achievable through a single-step reaction and the absence of interference with endogenous enzymes inherited from the extract. Consequently, this configuration establishes a straightforward yet robust system well-suited for various micro-compartment environments.

As indicated in the represented results, we demonstrated successful protein expression in three distinct microcompartments: water-in-oil droplets stabilized by semi-fluorinated oil, GUVs formed through emulsion transfer, and atop a supported lipid bilayer. The versatility of protein expression across different micro-compartments highlights the robustness of our CFPS system for synthetic cell construction. Among these encapsulation methods, the emulsion droplet technique was a notably straightforward approach for encapsulating the CFPS system, requiring only agitation or vortexing. However, these droplets tend to be unstable due to precarious water interface, a limitation that could be addressed by adding lipids or surfactants into the oil phase. Despite the practicality of stabilized water-in-oil droplets, they do not fully replicate the natural cell environment, which consists of two aqueous compartments separated by a cell membrane. Instead of droplets, giant unilamellar vesicles (GUVs) with sizes exceeding 1 micron were established to mimic cells more closely. The CFPS system was encapsulated within the lipid bilayer of these vesicles, furnishing transcription and translation machinery for diverse synthetic cell systems⁵⁵.

Emulsion transfer, also known as double emulsion, stands out as the typical technique for encapsulating CFPS reactions, owing to its mild preparation process and relatively high encapsulation efficiency compared to solvent-free methods. However, the generation of double emulsion vesicles can range from simple centrifugation steps to the use of microfluidic-assisted emulsion transfer, each method with its associated advantages and disadvantages, as summarized elsewhere³⁶. The successful encapsulation of the CFPS system within GUVs relies on tuning osmolarity and applying the proper force to traverse the oil-water interface. Although the emulsion transfer method described here is the most straightforward and requires no specific lab equipment, the efficiency and homogeneity of generated GUVs could vary between different batches. Moreover, all emulsion transfer methods must deal with residual oil in the lumen of the formed bilayer interface, necessitating an additional oil removal step⁵⁶, which may pose challenges in kinetic projects.

Finally, we have shown the successful expression of CFPS atop a Supported Lipid Bilayer (SLB), offering a valuable approach for projects requiring protein synthesis in proximity to the lipid biomembrane environment. Through the utilization of the presented CFPS system, we have demonstrated its successful application in characterizing the reversible membrane targeting process of Cdc42⁴⁷, a central protein molecule crucial for cellular polarity. In summary, the consistent medium expression yields and inherent robustness observed in various micro-compartments highlight the potential of this CFPS system in the construction of diverse synthetic cell systems. This capability can be further extended to include various protein-centered models, ultimately advancing towards the realization of minimal cells.

Materials

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)	Avanti	850375P
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)(DOPS)	Avanti	840035P
1,4 dithiothreitol (DTT)	Sigma-Aldrich	1.11474
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)	Avanti	850457P
3,5-cyclic AMP (cAMP)	Sigma-Aldrich	A9501
50 mL tubes	Eppendorf	Eppendorf Tubes BioBased
50% hydrogen peroxide	Sigma-Aldrich	516813
Acetate	Sigma-Aldrich	A6283
Agar powder	Sigma-Aldrich	05040
Alanin	Sigma-Aldrich	A4349
Amicon Stirred Cells	MerckMillipore	UFSC05001
Ammonium acetate (NH4OAc)	Sigma-Aldrich	A7262
Arginin	Sigma-Aldrich	A4474
Asparagin	Sigma-Aldrich	A0884
Aspartat	Sigma-Aldrich	A5474
ATP	Roche	11140965001
Atto 488 DOPE	Sigma-Aldrich	67335
Atto 647N DOPE	Sigma-Aldrich	42247
Baffled Erlenmeyer flask	Shuniu	250 mL, 1000mL
Bovine Serum Albumin(BSA)	Roche	10711454001
Centrifugetube	Eppendorf	Eppendorf Tubes 3810X
Centrifugetube rack	Eppendorf	0030119819
Chemiluminescence and epifluorescence imaging system	Uvitec	Alliance Q9 Advanced
Chloroform	Sigma-Aldrich	288306
Confocal Laser Scanning Microscopy (LSM)	ZEISS	LSM 780
Countess Cell Counting Chamber Slides	Thermo Fisher Scientific	C10283
Coverslip	Thermo Scientific	Menzel BB02400500A113MNZ0
creatine kinase (CK)	Roche	10127566001
Creatine phosphate (CP)	Sigma-Aldrich	10621714001
Culture dish	Huanqiu	90 mm
Cystein	Sigma-Aldrich	C5360
Cytidine 5'-triphosphate disodium salt (CTP)	aladdin	C101487
Dialysis membrane	Spectrum	Standard RC Tubing MWCO: 12-14 kD
E.Z.N.A. Cycle Pure Kit	Omega Bio-Tek	D6492-01
Electro-Heating Standing-Temperature Cultivator	Yiheng instrument	DHP-9602
Ethylenediaminetetraacetic acid(EDTA)	Biosharp	1100027
Fluorescent plate reader	BioTek	Synergy 2
Fluorinated oil	Suzhou CChip scientific instrument	2%HFE7500
Folinic acid	Sigma-Aldrich	47612
French Press	G.Heinemann	HTU-DIGI-Press
Glucose	Sigma-Aldrich	G7021
Glutamat	Sigma-Aldrich	G5667
Glutamin	Sigma-Aldrich	G5792
Glycerol	Sigma-Aldrich	G5516
Glycin	Sigma-Aldrich	G7126
Guanosine 5'-triphosphate sodium salt hydrate(GTP)	Roche	10106399001
HEPES	Sigma-Aldrich	H3375
HiPrep Q FF 16/10	Cytiva	28936543
Histidin	Sigma-Aldrich	H6034
Isoleucin	Sigma-Aldrich	I5281
Isopropyl-β-D-thiogalactopyranoside (IPTG)	Sigma-Aldrich	I5502
K2HPO4	Sigma-Aldrich	P8281
KH2PO4	Sigma-Aldrich	P5655
Leucin	Sigma-Aldrich	L6914
Lysin	Sigma-Aldrich	L5501
Magnesium acetate tetrahydrate (Mg(OAc)2 )	Sigma-Aldrich	M5661
Magnesium chloride(MgCl2)	Sigma-Aldrich	M2670
Methionin	Sigma-Aldrich	M8439
Microcentrifuge	Eppendorf	5424 R
Mineral oil	Sigma-Aldrich	M5904
Mini-PROTEAN Tetra Cell Systems	Bio-Rad	1645050
Multipurpose Centrifuge	Eppendorf	5810 R
NaN3	Sigma-Aldrich	S2002
Nucleic Acid & Protein UV-Assay Measurements	IMPLEN	NanoPhotometer N60
NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA	MACHEREY-NAGEL	740414.5
Nunc-Immuno MicroWell 96 well polystyrene plates	Sigma-Aldrich	P8616
PCR Thermal Cycler	Eppendorf	Mastercycler nexus
Peptone	Sigma-Aldrich	83059
Phenylalanin	Sigma-Aldrich	P8740
Phosphoenolpyruvat (PEP)	GLPBIO	GC44635
PMSF	Sigma-Aldrich	PMSF-RO
Polyethylene glycol 8000 (PEG 8000)	Sigma-Aldrich	89510
Potassium Acetate(KOAc)	Sigma-Aldrich	P5708
Potassium chloride(KCl)	Sigma-Aldrich	P9541
Potassium glutamate (K-glutamate)	Sigma-Aldrich	G1501
Potassium hydroxide(KOH)	Sigma-Aldrich	221473
Prolin	Sigma-Aldrich	P8865
Pyruvate kinase (PK)	Sigma-Aldrich	P9136
Serin	Sigma-Aldrich	S4311
Shaker	Zhichushakers	ZQZY-AF8
Sodium chloride(NaCl)	Sigma-Aldrich	S5886
Sodium hydroxide(NaOH)	Sigma-Aldrich	S5881
Sucrose	aladdin	S112226
Sulfuric acid	Sigma-Aldrich	339741
Syringe Filters	Jinteng	0.45 μm
Test tube	Shuniu	20 mL
TGX FastCast Acrylamide Kit, 12%	Bio-Rad	#1610175
ThermoMixer	Eppendorf	ThermoMixer C
Threonin	Sigma-Aldrich	T8441
Tris base	Sigma-Aldrich	V900483
tRNA	Roche	10109550001
Tryptone	Sigma-Aldrich	T7293
Tryptophan	Sigma-Aldrich	T8941
Tyrosin	Sigma-Aldrich	T8566
UTP Trisodium salt (UTP)	aladdin	U100365
Vacuum Pump with Circulated Water System	Zhengzhou Greatwall Scientific Industrial and Trade Co.Ltd	SHB-
Valin	Sigma-Aldrich	V4638
Vortex Mixers	Kylin-Bell	Vortex QL-861
Water purification system	MerckMillipore	Direct ultrapure water (Type 1)
Yeast extract	Sigma-Aldrich	70161
β-mercaptoethanol	Sigma-Aldrich	444203