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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#196;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&#160;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 &#38; 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/&#181;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&#160;&#916;&#160;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&#160;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>&#946;-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/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/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/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/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 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/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/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/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/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/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/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 ...

Research

JoVE Journal

Bioengineering

8.0K 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

Sealable Femtoliter Chamber Arrays for Cell-free Biology

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

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

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

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

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

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

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

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

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

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

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

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

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

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

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

Rapid Characterization of Genetic Parts with Cell-Free Systems

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

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

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

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

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

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

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

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

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

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

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

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.