Name: OnePot PURE Cell-Free System (Video) | JoVE Journal
Uploaded: 2021-06-23T13:00:00.000+00:00
Duration: 8 min 25 s
Description: Learn the OnePot PURE method for rapid cell-free protein expression using recombinant TX-TL systems. This protocol reduces preparation time from weeks to three days, enabling accessible synthetic biology research.

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This work was supported by the European Research Council under the European Union&#39;s Horizon 2020 Research and Innovation Program Grant 723106, a Swiss National Science Foundation Grant (182019), and EPFL.

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The authors declare that they have no competing financial interests.

The protocol presented here describes a simple, time- and cost-effective method to prepare a versatile PURE expression system20 based on the standard composition15. By utilizing the protocol together with the supplied daily schedules (Table 1), all components can be prepared in 1 week and yield amounts sufficient for up to five hundred 10 &#181;L PURE reactions. Since the proteins used in this protocol are overexpressed from high copy plasmids and have low toxicity to E. coli, good expression levels are observed for all the required proteins (Figure 1). This allows for the easy adjustment of strains, and therefore also protein composition in cocultures, simply by modifying the ratios of the inoculation strains20. Besides the ribosomal proteins, the concentration of EF-Tu showed to be of fundamental importance for expression yields6. In contrast, changes in the concentration of the other protein components had a relatively low impact on the robustness of the PURE system7,24. Therefore, by adjusting the inoculation ratio of EF-Tu with regard to all the other components, a comparable composition to the standard PURE composition can be achieved, and a PURE system with a similar yield20 can be attained. In preparing the protein solution, it is crucial to ensure that all strains grow well and overexpress the encoded protein after induction (Figure 1).
Ribosome function is key for the overall performance of the PURE system24. In this protocol, two different methods for preparing the ribosome solution are demonstrated, i.e., tag-free and His-tagged ribosome purification. The tag-free ribosome purification is based on hydrophobic interaction chromatography followed by centrifugation with a sucrose cushion, which requires access to a FPLC purification system and an ultracentrifuge15. In contrast, the method utilizing His-tagged ribosomes18 and gravity flow affinity chromatography purification does not require specialized equipment and can be performed in most laboratories. The latter method, therefore, brings advantages such as simplicity and accessibility. However, we observed a significantly lower synthesis yield when using the His-tagged ribosomes in the OnePot PURE compared to the tag-free variant (Figure 3). Based on the type of application, this lower yield may be acceptable.
The energy solution provides the low molecular weight components and tRNAs required to fuel in vitro TX-TL reactions. This protocol provides a recipe for a typical energy solution, which can be easily adjusted based on user needs. Together with tRNA, NTP, and creatine phosphate, the abundance and concentration of Mg2+ ions have been crucial for the overall performance of the PURE system8, as they are critical cofactors for transcription and translation. In some cases, the titration of ions can, therefore, greatly enhance the overall PURE performance. DNA integrity is crucial for PURE performance. Thus, sequence verifying the promoter region, ribosome binding site, and target gene and ensuring that an adequate DNA concentration (&#60;2 nM) will help troubleshoot issues that may arise while setting up a PURE reaction.
The PURE system is a minimal TX-TL system, and specific applications may thus require additional adjustments25. These may include incorporating different RNA polymerases9,26, chaperones13, and protein factors such as EF-P or ArfA8. Although the expression strains for these proteins can be included in the cocultures, adding them separately to the prepared system may provide better control of the required protein levels. Furthermore, the inclusion of vesicles is essential to the production of membrane proteins10,11. Oxidizing rather than reducing environments and a disulfide bond isomerase facilitate proper disulfide bond formation, which are, for example, required for secretory proteins12.
It is essential to ensure that any additional components do not interfere with the reaction. The most important factors to pay attention to when setting up a reaction or adding other components are listed below. Ensure that neither incompatible buffers are used nor the ion concentrations are disturbed. Avoid solutions containing glycerol, high concentrations of potassium, magnesium, calcium ions, osmolytes, pyrophosphate, antibiotics, or EDTA, as much as possible. For example, replacing an elution buffer with water during DNA purification can be beneficial as EDTA is a common additive in this buffer. Supplying the solutions with additional negatively charged molecules such as NTP or dNTP requires adjusting the magnesium concentration8, as the negatively charged molecules behave as chelating agents and bind positively charged molecules. A neutral pH is ideal for the reaction. Accordingly, all components should be buffered to the corresponding pH; this is especially important for highly acidic or basic molecules such as NTPs. Lastly, temperature and volume are key parameters for the reaction. To achieve a good yield, one should implement a temperature around 37 &#176;C, as temperatures below 34 &#176;C will significantly reduce the yield27.
It is relevant to note that before preparing the OnePot PURE, one should consider the target application and the associated requirements, such as volume, purity, ease of modification, and inclusion or omission of components. For many applications, the system will be an excellent choice, but others may require yields, adjustability, and other factors, which the OnePot system cannot provide. Irrespectively, the introduced protocol will be beneficial for the preparation of any home-made system, as all critical steps for such preparation are summarized here.
One of the main advantages of the OnePot system is its compatibility with the commercially available PURExpress system, which provides the possibility of testing the functionality and integrity of all components separately by sequentially replacing each PURExpress component with its OnePot equivalent. The advantages of the OnePot PURE system, such as tunability and easy, fast, and cost-effective preparation, will make cell-free TX-TL accessible to more laboratories worldwide and contribute to expanding the implementation of this powerful platform in cell-free synthetic biology.

Cell-free transcription-translation (TX-TL) systems constitute a promising platform for investigating and engineering biological systems. They provide simplified and tunable reaction conditions, as they no longer rely on life-sustaining processes, including growth, homeostasis, or regulatory mechanisms1. Thus, it is anticipated that cell-free systems will contribute to the investigation of biomolecular systems, offer a framework to test rational biodesign strategies2, and provide a chassis for a future synthetic cell3,4. The fully recombinant PURE system offers an especially appealing chassis due to its defined and minimal composition, as well as its adjustability and tuneability5.
Since the first functional, fully recombinant PURE system was established in 20015, efforts have been made to expand the system limits and optimize the system&#39;s composition to improve the system yields6,7,8, allow for transcriptional regulation9, membrane10,11 and secretory protein synthesis12, and to facilitate protein folding13,14. Nowadays, there are three commercially available systems: PUREfrex (GeneFrontier), PURExpress (NEB), and Magic PURE (Creative Biolabs). However, those systems are costly, their exact composition is proprietary and thus unknown, and adaptability is limited.
PURE systems prepared in-house proved to be the most cost-effective and tunable option15,16. However, the required 37 purification steps for protein and ribosome fractions are time-consuming and tedious. Several attempts have been made to improve the efficiency of the PURE system preparation17,18,19. We recently demonstrated that it is possible to coculture and co-purify all required non-ribosomal proteins present in the PURE system. This OnePot method has proved to be cost-effective and time-efficient, cutting down preparation time from several weeks to 3 working days. The approach generates a PURE system with a protein production capacity comparable to the commercially available PURExpress system20. Contrary to the previous approaches to simplify the PURE preparation17,18,19, in the OnePot approach all proteins are still expressed in separate strains. This enables the user to tune the composition of the OnePot PURE system by merely omitting or adding specific strains or adjusting the inoculation volumes, thus generating dropout PURE systems or altering the final protein ratios, respectively.
The protocol presented here provides a detailed method for creating the OnePot PURE system as described previously20, although &#946;-mercaptoethanol was replaced with tris(2-carboxyethyl)phosphine (TCEP). Moreover, two methods for ribosome purification are described: traditional tag-free ribosome purification using hydrophobic interaction and sucrose cushion, adapted from Shimizu et al.15, and Ni-NTA ribosome purification based on Wang et al.18 and Ederth et al.21 but significantly modified. The latter method further facilitates the preparation of the PURE system and makes it accessible to more laboratories, as only standard laboratory equipment is required.
The experimental protocol summarizes the preparation of a versatile PURE cell-free TX-TL system to provide a simple, tunable, cost-effective cell-free platform, which can be prepared using standard laboratory equipment within a week. Besides introducing the standard PURE composition, we indicate how and where it can be adjusted, with a primary focus on critical steps in the protocol to ensure the system&#39;s functionality.

<table><tbody><tr><td>10x Tris/Glycine/SDS buffer</td><td>Bio-Rad Laboratories</td><td>1610732</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>VWR International</td><td>525-0309</td><td></td></tr><tr><td>384-well Black Assay Plates</td><td>Corning</td><td>3544</td><td></td></tr><tr><td>4-20% Mini-PROTEANRTM TGXTM Precast Protein Gels</td><td>Bio-Rad Laboratories</td><td>4561096</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>VWR International</td><td>525-0304</td><td></td></tr><tr><td>96-Well Polypropylene DeepWell plate</td><td>Nunc</td><td>260252</td><td></td></tr><tr><td>Acetic acid, 99.8 %</td><td>Acros</td><td>222140010</td><td></td></tr><tr><td>&amp;Auml;kta purifier</td><td>GE Healthcare</td><td></td><td>purification of tag free ribosomes</td></tr><tr><td>AMICON ULTRA 0.5 mL - 3 KDa</td><td>Merck Millipore</td><td>UFC500324</td><td></td></tr><tr><td>AMICON ULTRA 15 mL - 3 KDa</td><td>Merck Millipore</td><td>UFC900324</td><td></td></tr><tr><td>Amino acids</td><td>Sigma-Aldrich</td><td>LAA21-1KT</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>09718-250G</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma-Aldrich</td><td>A4418</td><td></td></tr><tr><td>Ampicillin</td><td>Condalab</td><td>6801</td><td></td></tr><tr><td>BenchMark Fluorescent Protein Standard</td><td>ThermoFisher</td><td>LC5928</td><td></td></tr><tr><td>Breathe-Easy&amp;nbsp;sealing membrane</td><td>Diversified Biotech</td><td>Z380059-1PAK</td><td></td></tr><tr><td>Centrifuge tubes polycarbonate</td><td>Beckman</td><td>355631</td><td>purification of tag free ribosomes</td></tr><tr><td>Chill-out Liquid Wax</td><td>Bio-Rad Laboratories</td><td>CHO1411</td><td></td></tr><tr><td>Creatine phosphate</td><td>Sigma-Aldrich</td><td>27920</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator-25 (Capped)</td><td>Zymo</td><td>ZYM-D4034-200TS</td><td></td></tr><tr><td>DTT</td><td>SantaCruz Biotech</td><td>sc-29089B</td><td></td></tr><tr><td>Econo-Pac Chromatography Columns</td><td>Bio-Rad Laboratories</td><td>7321010</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Sigma-Aldrich</td><td>03609-250G</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes</td><td>VWR International / Eppendorf</td><td>525-0133</td><td></td></tr><tr><td>Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap</td><td>Falcon</td><td>352051</td><td></td></tr><tr><td>Flasks, baffled 1000 mL 4 baffles, borosilicate glass</td><td>Scilabware</td><td>9141173</td><td></td></tr><tr><td>FluoroTect Green Lys in vitro Translation Labeling System</td><td>Promega</td><td>L5001</td><td>optional</td></tr><tr><td>Folinic acid</td><td>Sigma-Aldrich</td><td>PHR1541</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7757-1L</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>HiTrap Butyl HP Column</td><td>GE Healthcare</td><td>28411005</td><td>purification of tag free ribosomes</td></tr><tr><td>IMAC Sepharose 6 Fast Flow</td><td>GE Healthcare</td><td>17-0921-07</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>InstantBlue</td><td>Expedeon</td><td>ISB1L-1L</td><td></td></tr><tr><td>IPTG (Isopropyl-beta-D-thiogalactoside)</td><td>Alfa Aesar</td><td>B21149.03</td><td></td></tr><tr><td>Laemmli buffer (2x), sample buffer</td><td>Sigma-Aldrich</td><td>S3401-1VL</td><td></td></tr><tr><td>Lysogeny broth (LB) media</td><td>AppliChem</td><td>A0954</td><td></td></tr><tr><td>Magnesium acetate</td><td>Sigma-Aldrich</td><td>M0631</td><td></td></tr><tr><td>Magnesium chloride</td><td>Honeywell Fluka</td><td>63020-1L</td><td></td></tr><tr><td>Nickel Sulfate</td><td>Alfa Aesar</td><td>15414469</td><td></td></tr><tr><td>NTP</td><td>ThermoFisher</td><td>R0481</td><td></td></tr><tr><td>Phusion High-Fidelity DNA Polymerase (2 U/&amp;micro;L)</td><td>ThermoFisher</td><td>F530S</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P5405-1KG</td><td></td></tr><tr><td>Potassium glutamate</td><td>Sigma-Aldrich</td><td>49601</td><td></td></tr><tr><td>PURExpress In Vitro Protein Synthesis Kit</td><td>NEB</td><td>E6800S</td><td></td></tr><tr><td>PURExpress&amp;nbsp;&amp;Delta;&amp;nbsp;Ribosome Kit</td><td>NEB</td><td>E3313S</td><td></td></tr><tr><td>Quick Start Bradford 1x Dye Reagent</td><td>Bio-Rad Laboratories</td><td>5000205</td><td></td></tr><tr><td>Rapid-Flow Sterile Single Use Vacuum Filter Units</td><td>ThermoFisher</td><td>564-0020</td><td></td></tr><tr><td>RNaseA solution</td><td>Promega</td><td>A7973</td><td></td></tr><tr><td>SealPlate&amp;nbsp;film</td><td>Excel Scientific</td><td>Z369659-100EA</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>6203</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>S2626</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>TCEP (Tris(2-carboxyethyl)phosphin -hydrochlorid)</td><td>Sigma-Aldrich</td><td>646547-10X1mL</td><td></td></tr><tr><td>Thickwall Polycarbonate Tube</td><td>Beckman</td><td>355631</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>T0699</td><td></td></tr><tr><td>Tris base</td><td>ThermoFisher</td><td>BP152-500</td><td></td></tr><tr><td>tRNA</td><td>Roche</td><td>10109541001</td><td></td></tr><tr><td>Ultracentrifuge Optima L-80</td><td>Beckman</td><td></td><td>purification of tag free ribosomes</td></tr><tr><td>Whatman GD/X syringe filters</td><td>GE Whatman</td><td>WHA68722504</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250-100mL</td><td></td></tr></tbody></table>

onepot pure cell-free system

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.

The above protocol is designed to facilitate establishing the PURE cell-free TX-TL system in any laboratory. The protocol includes a detailed description of the preparation of the three distinct parts of the PURE system: the OnePot protein, ribosome, and energy solution. A detailed daily schedule, which optimizes the workflow, is shown in Table 1. The workflow is optimized for the purification of His-tagged ribosomes, and time frames may differ slightly if tag-free ribosome purification is performed. One preparation provides a sufficient amount of PURE for a minimum of five hundred 10 µL reactions. Moreover, the prepared solutions are stable for more than a year at -80 °C and can withstand multiple freeze-thaw cycles.
Adequate overexpression levels for all strains are crucial for the functionality of the final protein solution. Figure 1 shows successful overexpression in all 36 individual strains used subsequently for the OnePot protein preparation. Variation in the over-expressed proteins' band intensities occurred most probably due to a bias in loading volumes onto the SDS-PAGE gel. The expected protein sizes are summarized in Table 2. GlyRS and PheRS consist of two subunits of various molecular weights; the remaining 34 proteins consist of a single subunit. Key to this protocol's simplicity and time-effectiveness is the coculturing and co-purification step (Figure 2). The OnePot protein solution was prepared by increasing the ratio of EF-Tu strain with respect to all the other expression strains. The overall composition of the final proteins was analyzed by SDS-PAGE (Figure 3A). From the gels (lanes 2, 3), it is noticeable that EF-Tu (43.3 kDa) is present in a higher concentration compared to the other proteins, as expected. While the gel provides a good first indication of protein expression ratios, it is difficult to determine whether and at which level each individual protein was expressed. Therefore, it is highly recommended to confirm the overexpression in each strain before coculturing, as shown above.
The E. coli ribosome is a complex molecular machine composed of over 50 individual protein subunits23. A representative absorption spectrum at 260 nm for tag-free ribosome purification is shown in Figure 4; the third peak is characteristic of successful ribosome elution. For both ribosome purification methods, the expected running pattern on the SDS-PAGE gel (Figure 3A)18 was observed. We did observe contaminations for both purifications, albeit in small quantities (&lt;10%). Notably, different contaminants were present in the tag-free (lanes 5, 6) and His-tagged (lanes 11, 12) ribosomes due to the variation in the method. For user reference, the SDS-PAGE gels for the combined systems are also included (lanes 8, 9, and 14, 15).
Lastly, the performance of the prepared systems (Figure 3) using the different ribosome variants are compared. The time courses of in vitro eGFP expression show that both PURE systems are functional and produce fluorescent eGFP. However, the OnePot protein solution combined with the His-tagged ribosomes, using the ribosome concentration optimized by titration, yielded only one-third of the expression level of the non-tagged ribosome version (Figure 3B). Similar results were observed when three proteins of different sizes were expressed and labeled using the Green Lys tRNA in vitro labeling system (Figure 3C). As seen on the fluorescent gel, full-length products were successfully expressed in both systems; however, only around half of the expression level was achieved with the His-tag ribosome system. In addition to the fluorescence labeling, the expected bands for all three proteins are distinguishable on a Coomassie-stained gel (Figure 3D). The results show that the introduced expression system, which can be prepared within a week in a laboratory with standard equipment, can be used for the in vitro expression of proteins encoded downstream of the T7 promoter from linear templates.
<img alt="SDS-PAGE protein expression test, gel electrophoresis results for analyzing protein bands." class="xfigimg" src="/files/ftp_upload/62625/62625fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Representative results for the overexpression test for all expression strains of the PURE system. PURE protein numbers and sizes are summarized in Table 2. Protein numbers 21, 24, and 27 are marked with a star for better visualization. <a href="https://www.jove.com/files/ftp_upload/62625/62625fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="OnePot protein purification process diagram; resuspension, sonication, incubation, purification steps." class="xfigimg" src="/files/ftp_upload/62625/62625fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: OnePot protein purification. The schematic depiction and corresponding photographs of all steps involved in the production of the OnePot protein solution. <a href="https://www.jove.com/files/ftp_upload/62625/62625fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE protein analysis; eGFP expression; ribosome performance graph; tag-free vs His-tag ribosomes." class="xfigimg" src="/files/ftp_upload/62625/62625fig03.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: Performance of the prepared systems using the different ribosome variants. (A) Coomassie blue stained SDS-PAGE gels of the OnePot protein solution (lanes 2, 3), tag-free ribosomes without protein solution (lanes 5, 6) and with protein solution (lanes 8, 9), His-tagged ribosomes without protein solution (lanes 11, 12) and with protein solution (lanes 14, 15). Two different concentrations were loaded per sample. (B) Comparison of eGFP expression of His-tagged ribosomes and tag-free ribosomes. The fluorescence intensity of in vitro eGFP expression is monitored over time for a PURE reaction using tag-free ribosomes (1.8 µM, blue) and His-tagged ribosomes (0.62 µM, red). The concentrations of the linear template and the OnePot protein solution were 4 nM and 2 mg/mL, respectively. Panels (C) and (D) show the SDS-PAGE gel of proteins synthesized in OnePot with tag-free (1.8 µM, blue, lanes 3, 4, 5) and His-tag ribosomes (0.62 µM, red, lanes 6, 7, 8) labeled with a GreenLys in vitro labeling kit (C) and stained with Coomassie blue (D), respectively. The black arrows indicate the expected bands of synthesized proteins: eGFP (26.9 kDa), ArgRS (64.7 kDa), T7 RNAP (98.9 kDa). The linear template and OnePot protein solution concentrations were 4 nM and 1.6 mg/mL, respectively. <a href="https://www.jove.com/files/ftp_upload/62625/62625fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography result; absorbance vs. volume; elution peaks; protein purification graph." class="xfigimg" src="/files/ftp_upload/62625/62625fig04.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
Figure 4: Absorbance spectra at 260 nm. Representative results of absorbance spectra at 260 nm during hydrophobic interaction purification of tag-free ribosomes. <a href="https://www.jove.com/files/ftp_upload/62625/62625fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: A daily time-optimized schedule for the preparation of all the OnePot PURE solutions. <a href="https://www.jove.com/files/ftp_upload/62625/Table1.pdf" target="_blank">Please click here to download this Table.</a>
Table 2: PURE protein list <a href="https://www.jove.com/files/ftp_upload/62625/Table2.pdf" target="_blank">Please click here to download this Table.</a>
Supplementary Table 1: Reagents. The table lists concentrations, volumes, and other specific details of the reagents and components used during this study. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%201-62625.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 2: Buffers. The spreadsheet lists the exact buffer compositions for protein, tag-free ribosome, and His-tag ribosome purifications, as well as the concentrations of the stock solutions used for their preparation. In addition, it calculates the required amounts of components based on the buffer volume. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%202_.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 3: Amino acid calculations. The spreadsheet lists the amino acids and their recommended stock solution concentrations required for the energy solution. It calculates the amount of water to be added to each amino acid based on the actual weighed mass, and also calculates the volume of the amino acid solution to be added to the final amino acids' mixture. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%203-62625.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 4: Stock solutions for the energy solution. The table lists the concentrations and volumes of stock solutions needed for the energy solution and indicates further details, including storage conditions. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%204_.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 5: Energy solution. The table lists the energy solution components and their recommended concentrations. In addition, it calculates their required volumes to be added to the final solution based on their stock solution concentrations and the volume of the energy solution. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%205-62625.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 6: PCR. The table lists sequences and concentrations of the primers used for the extension PCR and indicates melting temperatures and thermocycler steps optimized for a high-fidelity DNA polymerase. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Table%206-62625.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 7: PURE reaction. The spreadsheet shows an example setup of a PURE reaction. It lists the used concentrations and volumes of the components for a PURE reaction using tag-free ribosomes or His-tag ribosomes. Moreover, it calculates the volume ratios for protein and ribosome titrations. <a href="https://www.jove.com/files/ftp_upload/62625/Supplementary_Tables%207-62625.xlsx" target="_blank">Please click here to download this Table.</a>

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 ...

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