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

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 &#181;L reactions. Moreover, the prepared solutions are stable for more than a year at -80&#160;&#176;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&#39; 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&#39;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 (&#60;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="Figure 1" class="xfigimg" src="/files/ftp_upload/62625/62625fig01.jpg" /> 
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.bdigitaluss.remotexs.co/files/ftp_upload/62625/62625fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62625/62625fig02.jpg" /> 
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.bdigitaluss.remotexs.co/files/ftp_upload/62625/62625fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62625/62625fig03.jpg" /> 
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 &#181;M, blue) and His-tagged ribosomes (0.62 &#181;M, 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 &#181;M, blue, lanes 3, 4, 5) and His-tag ribosomes (0.62 &#181;M, 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.bdigitaluss.remotexs.co/files/ftp_upload/62625/62625fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62625/62625fig04.jpg" /> 
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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 1-62625.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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 2_.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&#39; mixture. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 3-62625.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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 4_.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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 5-62625.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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Table 6-62625.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.bdigitaluss.remotexs.co/files/ftp_upload/62625/Supplementary_Tables 7-62625.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about OnePot PURE Cell-Free System at JoVE.com

OnePot PURE Cell-Free System

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 ...

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.4K Views.  École Polytechnique Fédérale de Lausanne. We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

Video: OnePot PURE Cell-Free System

Bacterial Detection &amp; Identification Using Electrochemical Sensors

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

Bacterial Detection & Identification Using Electrochemical Sensors

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

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

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

Genetics

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

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

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

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

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.

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 ...

Biochemistry

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 ...

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 ...

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 ...

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 ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Biology

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, ...

OnePot PURE Cell-Free System

Summary

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Chapters in this video

Bacterial Detection & Identification Using Electrochemical Sensors

Reusable Single Cell for Iterative Epigenomic Analyses

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

Cell-Free Protein Synthesis System for Building Synthetic Cells

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

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

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

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

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate