Name: Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis (Video) | JoVE Journal
Uploaded: 2025-08-22T13:45:00.000+00:00
Duration: 8 min 26 s
Description: Learn to refine the OnePOT PURE system and crude ribosome preparation for reproducible cell-free protein synthesis. This protocol standardizes growth conditions and glycerol use to improve yields and accessibility for synthetic biology research.

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5 minutes

NL, SL, AB, and NZ are supported by NL&#39;s UKRI Future Leaders Fellowship (MR/V027107/1). SL is additionally supported by Biophoundry Inc. SY is supported by a Darwin Trust PhD studentship from the University of Edinburgh. CW is supported by a BBSRC Eastbio PhD studentship. The authors thank A. Pompa for assistance in using the ultracentrifuge, which was supported by funding from the Wellcome Discovery Research Platform for Hidden Cell Biology [226791]. Additionally, we thank B. Lavickova and S. J. Maerkl for assistance in setting up the original OnePot protocol. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising from this submission. Figure 1 was partially created in BioRender. Laohakunakorn, N. (2025) [https://BioRender.com/wj65i81].

<ol><li>Laohakunakorn, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bottom-up+construction+of+complex+biomolecular+systems+with+cell-free+synthetic+biology%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">Bottom-up construction of complex biomolecular systems with cell-free synthetic biology.</a> Front Bioeng Biotechnol. 8 (213), 1-26 (2020).</li>
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<li>Shepherd, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+design+and+synthesis+of+a+30-cistron+translation-factor+module%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">De novo design and synthesis of a 30-cistron translation-factor module.</a> Nucleic Acids Res. 45 (18), 10895-10905 (2017).</li>
<li>Villarreal, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+microbial+consortia+enable+rapid+assembly+of+pure+translation+machinery%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Synthetic microbial consortia enable rapid assembly of pure translation machinery.</a> Nat Chem Biol. 14 (1), 29-35 (2018).</li>
<li>Lavickova, B., Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple%2C+robust%2C+and+low-cost+method+to+produce+the+PURE+cell-free+system%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">A simple, robust, and low-cost method to produce the PURE cell-free system.</a> ACS Synth Biol. 8 (2), 455-462 (2019).</li>
<li>Grasemann, L., Lavickova, B., Elizondo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OnePot+PURE+cell-free+system%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">OnePot PURE cell-free system.</a> J Vis Exp. (172), e62625(2021).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+protein+expression+in+the+One-Pot+PURE+system%3A+insights+into+reaction+composition+and+translation+efficiency%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">Optimizing protein expression in the One-Pot PURE system: insights into reaction composition and translation efficiency.</a> ACS Synth Biol. 14 (5), 1496-1508 (2025).</li>
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<li>Doerr, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+cell-free+RNA+and+protein+synthesis+with+minimal+systems%5BTitle%5D)+AND+%22Phys+Biol%22%5BJournal%5D)">Modelling cell-free RNA and protein synthesis with minimal systems.</a> Phys Biol. 16 (2), 025001(2019).</li>
<li>Christodoulou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heteronuclear+NMR+investigations+of+dynamic+regions+of+intact+Escherichia+coli+ribosomes%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes.</a> PNAS. 101 (30), 10949-10954 (2004).</li>
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<li>Laohakunakorn, N. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLaohakunakorn%2C+N+%22Cell-free+3PGA+energy+solution%22">Cell-free 3PGA energy solution</a>. , Protocols.io. (2020).</li>
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</ol>

SL is co-founder and CTO of Biophoundry Inc., which develops cell-free technology for phage engineering.

We have presented a detailed procedure that has allowed us to obtain reproducible PURE systems over multiple batches and users in the same lab. There are a number of critical steps required to achieve this.
The first important step is to ensure the integrity of the 36 PURE strains: in addition to sequencing all plasmids, it is essential to carry out an expression trial to verify all strains are overexpressing correctly, as the loss of a single strain can abolish CFPS activity. We have observed...

Cell-free protein synthesis (CFPS) has matured as a technology for diverse applications, including biomanufacturing, biosensing and diagnostics, prototyping of genetic and metabolic parts and pathways, rapid protein engineering, and education1,2. CFPS systems using purified components, originally developed as the &#39;Protein synthesis Using Recombinant Elements&#39; (PURE) system3, offer a number of advantages over their lysate-based alternatives. PURE systems have a defined composition, consisting of 36 protein factors, the 70S E. coli ribosome, and a defined mixture of chemical substrates including nucleoside triphosphates (NTPs), amino acids, and tRNAs, which can be tailored at will to the required application. They are thus highly suited to studies such as non-canonical amino acid incorporation, mechanistic modelling, and the bottom-up construction of biomolecular systems and synthetic cells1. While PURE is available from commercial suppliers, its cost ($0.34-$1.63 per 1 &#181;L reaction) is prohibitive to large-scale experimentation; and the alternative strategy of producing PURE in-house has so far been hampered by labor-intensive protocols involving parallel purification of 36 proteins and ribosomes4, which are challenging to carry out robustly and reproducibly, particularly for inexperienced users.
Recent developments have aided the homemade production of PURE. For example, Wang et al. have engineered strains of E. coli to include genomically integrated hexa-histidine tags on the translation machinery, facilitating a single-step purification5. Genes encoding all the PURE proteins have been assembled into a small number of plasmids, which have similarly been used to reduce the purification labor6. Villareal et al. have engineered elegant consortia of PURE strains, which balance expression in order to produce proteins at the correct ratio, while at the same time allowing for single-step purification7.
The innovative OnePot PURE system developed by Lavickova et al. holds great promise for ease of use8,9. In this system, the original PURE strains are co-cultured and purified via a single chromatography step. Although it is not possible to precisely define the composition of the resulting PURE system, the authors demonstrated that multiple batches of the system are largely reproducible, and a coarse level of control can be achieved by varying the inoculation volume of each strain. The tradeoff of precise compositional control for ease of production is one which will be acceptable to many users, given that the overall protein synthesis yield from the OnePot system is similar to that obtained commercially. However, while detailed protocols exist for the OnePot system, it has been challenging to completely reproduce the Lavickova et al. results in other labs. For example, expression vector instability, slow-growing strains, and proteolysis have been observed10.
In this work, we present a detailed and refined OnePot PURE protocol, which controls the cell growth by using standardized glycerol stocks and media adjustments, gives guidance on monitoring growth and harvest OD, and normalizes the amount of glycerol in the final purification. Secondly, we present an optimized crude ribosome purification procedure using differential ultracentrifugation only, which balances the tradeoff between ribosome activity, and the cost and complexity of more sophisticated methods.
Although this introduces more steps to the OnePot protocol, they allow the user increased opportunity to troubleshoot the procedure more thoroughly if required, as well as modify it to their needs. We demonstrate the reproducibility of the process by showing multiple biological replicates carried out by multiple users in the same laboratory. We find that control over both the PURE components and the ribosome preparation are crucial to ensuring high CFPS performance of the system.
The PURE and ribosome preparation each takes 4 or 5 days, respectively, and can be performed either sequentially by one user or concurrently by two users. The PURE preparation involves 4x500 mL of liquid culture, each yielding ~2 g of cell pellet and ~1 mL of PURE proteins at a concentration of ~15 mg/mL. The ribosome preparation involves 4 &#215; 500 mL = 2 L of liquid culture, leading to a total of ~3 g of cell pellet and yielding ~1 mL of crude ribosomes at a concentration of ~17 &#181;M. The current protocol produces PURE systems with synthesis yields of up to ~100 &#181;g/mL GFP or ~130 &#181;g/mL of mCherry over 2 h of batch reaction11, which compares favorably to the original OnePot as well as commercial systems (~140-160 &#181;g/mL)8,12. Hence, it is a suitable starting point for further optimization through supplementation or modulation of sensitive components such as salts, crowding agents, and individual protein factors.

<table><tbody><tr><td>2x YT</td><td>Sigma</td><td>Y2377-250G</td><td></td></tr><tr><td>amino acids</td><td>Sigma</td><td>LAA21-1KT</td><td></td></tr><tr><td>ammonium acetate RNAse-free</td><td>Thermo Scientific</td><td>AM9070G</td><td></td></tr><tr><td>ammonium chloride</td><td>Sigma</td><td>09718-1KG</td><td></td></tr><tr><td>ampicillin</td><td>Sigma</td><td>A8351-5G</td><td></td></tr><tr><td>BL21(DE3) competent E. coli</td><td>NEB</td><td>C2527H</td><td></td></tr><tr><td>bottle, centrifuge (Nalgene)</td><td>Thermo Scientific</td><td>3120-9500</td><td>polypropylene co-polymer bottle</td></tr><tr><td>bottle, glass (Duran) 250 mL, 500 mL, 1 L, 2 L</td><td>VWR</td><td>215-1593, 215-1594, 215-1595, 215-1596</td><td></td></tr><tr><td>Bradford assay, Quick Start 1x Dye Reagent</td><td>Bio-Rad</td><td>5000205</td><td></td></tr><tr><td>centrifuge</td><td>Thermo Scientific</td><td>Heraeus Megafuge 16R</td><td></td></tr><tr><td>centrifuge, micro</td><td>Eppendorf</td><td>5424R&amp;nbsp;</td><td></td></tr><tr><td>chromatography column</td><td>Bio-Rad</td><td>7321010</td><td></td></tr><tr><td>creatine phosphate</td><td>Sigma</td><td>27920</td><td></td></tr><tr><td>cuvettes, semi micro 1.6 mL</td><td>Fisherbrand</td><td>FB55147</td><td></td></tr><tr><td>di-sodium hydrogen orthophophosphate dihydrate</td><td>Fisher Chemical</td><td>S/4450/60</td><td></td></tr><tr><td>dialysis cassette, Slide-A-Lyzer G3, 2 kDa MWCO, 15 mL</td><td>Thermo Scientific</td><td>A52962</td><td></td></tr><tr><td>dipotassium hydrogen orthophosphate anhydrous</td><td>Fisher Chemical</td><td>P/5240/60</td><td></td></tr><tr><td>DTT No-Weigh</td><td>Thermo Scientific</td><td>A39255</td><td></td></tr><tr><td>filter unit, centrifugal 3kDa MWCO Amicon Ultra 15</td><td>EMD Millipore</td><td>UFC900324</td><td></td></tr><tr><td>flask, 500 mL</td><td>VWR</td><td>214-1133</td><td></td></tr><tr><td>flask, baffled 2.5 L&amp;nbsp;</td><td>Tunair</td><td>SS-5013</td><td></td></tr><tr><td>folinic acid</td><td>Sigma</td><td>F7878-100MG</td><td></td></tr><tr><td>glycerol</td><td>Sigma</td><td>G5516-1L</td><td></td></tr><tr><td>HCl</td><td>Fisherbrand</td><td>H/1200/PB17</td><td></td></tr><tr><td>HEPES buffer, 1 M solution pH 7.3</td><td>Thermo Scientific</td><td>BP299</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>I2399-500G</td><td></td></tr><tr><td>incubator</td><td>Eppendorf</td><td>New Brunswick Innova&amp;reg; 42</td><td></td></tr><tr><td>IPTG</td><td>Thermo Scientific</td><td>R0392</td><td></td></tr><tr><td>KOH pellets</td><td>Thermo Scientific</td><td>P1767-500G</td><td></td></tr><tr><td>L-Glutamic acid hemimagnesium salt tetrahydrate</td><td>Sigma</td><td>49605-250G</td><td></td></tr><tr><td>L-Glutamic acid monopotassium salt monohydrate</td><td>Sigma</td><td>49601-500G</td><td></td></tr><tr><td>magnesium acetate tetrahydrate</td><td>Sigma</td><td>M5661-50G</td><td></td></tr><tr><td>magnesium chloride</td><td>Fluka</td><td>63020-1L</td><td></td></tr><tr><td>magnetic stirring setup</td><td>Stuart</td><td>US152</td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>NEBExpress Ni-NTA Mag Beads</td><td>NEB</td><td>S1423S</td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7543-1EA</td><td></td></tr><tr><td>pipette, multichannel Eppendorf Research Plus 30-300 &amp;micro;L</td><td>Eppendorf</td><td>3125000052</td><td></td></tr><tr><td>plate adhesive seal for plate reader</td><td>ExcelScientific</td><td>STR-SEAL-PLT (Sterile)</td><td></td></tr><tr><td>plate breathable seal</td><td>Starlab</td><td>E2796-3005</td><td></td></tr><tr><td>plate foil seal</td><td>ExcelScientific</td><td>FCS-25</td><td></td></tr><tr><td>plate reader</td><td>BioTek</td><td>Synergy H1</td><td></td></tr><tr><td>plate shaker</td><td>Allsheng</td><td>MBI100-4A</td><td></td></tr><tr><td>plate, 384 well</td><td>Thermo Scientific</td><td>242764</td><td></td></tr><tr><td>plate, 96 deep well 1 mL</td><td>Thermo Scientific</td><td>736-0601</td><td></td></tr><tr><td>plate, 96 deep well 2 mL</td><td>Sarstedt</td><td>82.1972.002</td><td></td></tr><tr><td>plate, 96 well PCR</td><td>Sarstedt</td><td>721,978</td><td></td></tr><tr><td>plate, optical 96 well</td><td>Thermo Scientific</td><td>10057581</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma</td><td>P5405-500G</td><td></td></tr><tr><td>potassium dihydrogen orthophosphate</td><td>Fisher Chemical</td><td>P/4800/53</td><td></td></tr><tr><td>protein extraction (BugBuster) reagent</td><td>EMD Millipore</td><td>70584-3</td><td></td></tr><tr><td>protein purification resin (cOmplete)</td><td>Roche</td><td>5893682001</td><td></td></tr><tr><td>rotisserie shaker</td><td>Starlab</td><td>RM-Multi 1</td><td></td></tr><tr><td>sodium chloride</td><td>Fisher Chemical</td><td>S/3105/70</td><td></td></tr><tr><td>sonication probe, FisherBrand 15-50 mL</td><td>Thermo Scientific</td><td>12931181</td><td></td></tr><tr><td>sonicator</td><td>Fisher Scientific</td><td>FB120</td><td></td></tr><tr><td>spectrophotometer</td><td>Jenway</td><td>Genova Plus</td><td></td></tr><tr><td>spermidine</td><td>Sigma</td><td>S2626-1G</td><td></td></tr><tr><td>TCEP 0.5 M</td><td>Supelco</td><td>646547</td><td></td></tr><tr><td>Thermo Fisher NTP, Tris buffered</td><td>Thermo Scientific</td><td>R1481</td><td></td></tr><tr><td>tRNA from E. coli MRE600</td><td>Roche</td><td>10109541001</td><td></td></tr><tr><td>tryptone</td><td>Formedium</td><td>TRP02</td><td></td></tr><tr><td>tube, 0.2 mL PCR grade</td><td>Sarstedt</td><td>72,737,002</td><td></td></tr><tr><td>tube, bijoux Sterilin 7 mL polystyrene</td><td>Thermo Scientific</td><td>129A</td><td></td></tr><tr><td>tube, conical (Falcon) 15 mL</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>tube, conical (Falcon) 50 mL</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>tube, microcentrifuge (Eppendorf) 1.5 mL</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>tube, microcentrifuge (Eppendorf) 2 mL</td><td>Sarstedt</td><td>72.695.700</td><td></td></tr><tr><td>tube, S100 centrifugation&amp;nbsp;</td><td>Beckman-Coulter</td><td>355619</td><td></td></tr><tr><td>tube, S30 centrifugation&amp;nbsp;</td><td>Thermo Scientific</td><td>3118-0050</td><td></td></tr><tr><td>ultracentrifuge</td><td>Beckman-Coulter</td><td>Optima XPN-100</td><td></td></tr><tr><td>ultracentrifuge rotor</td><td>Beckman-Coulter</td><td>70Ti fixed angle rotor</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Thermo Scientific</td><td>10977049</td><td></td></tr><tr><td>vortexer</td><td>VWR</td><td>444-2791</td><td></td></tr><tr><td>yeast extract</td><td>Oxoid</td><td>LP0021T</td><td></td></tr></tbody></table>

refinement of onepot pure and crude ribosome production for reproducible cell-free protein synthesis

Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis

Recent advances have enabled the Protein synthesis Using Recombinant Elements (PURE) cell-free system to be produced in individual laboratories economically and with reduced labor burden. However, the preparation of the 36 protein components and ribosome, which make up PURE, is still a complex undertaking, with much scope for variation and error. We present a detailed and updated procedure to manufacture PURE based on the recently published OnePot protocol, which involves regulating a number of key steps, in particular, the inoculation of cultures using optical density (OD)-normalized glycerol stocks, careful monitoring of cell growth, and controlling final glycerol concentrations. Using this approach, we report reproducible preparations of PURE from multiple biological replicates and users. This protocol serves as a reference and starting point for other laboratories to manufacture and troubleshoot their own PURE systems, thus increasing democratization of the many applications that PURE enables, such as the bottom-up construction of biomolecular systems and the development of synthetic cells.

The workflow for the PURE and ribosome preparations is shown in Figure 1, and a recommended timetable for the work is shown in Table 1. Typically, reactions are monitored through the production of fluorescent proteins such as GFP or mCherry, and RFU readings can be converted to concentrations by calibration11. However, CFPS yields depend on the specific protein being produced, and so it is important to optimize the sys...

We present an updated protocol for the production of the OnePot 'Protein synthesis Using Recombinant Elements' (PURE) system and crude ribosomes for cell-free protein synthesis, focusing on controlling cell growth to minimize batch-to-batch variations.

Recent advances have enabled the Protein synthesis Using Recombinant Elements (PURE) cell-free system to be produced in individual laboratories ...

We refined the OnePOT PURE system and crude ribosome preparation to improve reproducibility and accessibility, enabling more labs to build and troubleshoot robust cell-free systems for synthetic biology research. Experimental challenges include batch-to-batch variability, expression vector instability, and slow-growing strains, all of which hinder reproducibility and consistent performance in preparing in-house PURE systems. We established that careful control of strain growth, inoculation, and glycerol concentration significantly improves reproducibility and yields in OnePOT PURE and ribosome preparations across users and biological replicates.We address the lack of a reproducible, accessible protocol for preparing PURE and ribosomes in house, helping labs overcome technical barriers to adopt self resistance for synthetic biology research. Our protocol improves reproducibility and flexibility by standardizing growth conditions and glycerol use and streamlines ribosome preparation using only ultracentrifugation, avoiding more complex or costly purification methods. To begin, thaw the frozen BL21 cell pellets on ice and add 50 milliliters of Ribosome Buffer A into a clean conical tube.Using a pipette, take 100 microliters of Ribosome Buffer A from the conical tube and add it to a No-Weigh DTT vial. Aspirate gently until the DTT pellet is completely dissolved to create a 0.5 molar DTT stock solution. Then, pipette 100 microliters of the 0.5 molar DTT stock solution back into the 50 milliliter conical tube and place the tube on ice.Using a vortex mixer, re-suspend each thawed cell pellet in Ribosome Buffer A with DTT at a ratio of two milliliters per one gram of cell pellet. Combine the resuspended samples into a conical tube and check that the total slurry volume is 10 milliliters. Now, divide the 10 milliliter slurry equally into two separate five-milliliter samples in labeled conical tubes.Secure the conical tubes in a stand and lyse the cells using a cooled sonication probe in an ice water bath. Set the sonicator to 30%amplitude with a cycle of 20 seconds on and 20 seconds off for a total of 10 cycles. After sonication, top up each lysed sample to 25 milliliters using Ribosome Buffer A with DTT and briefly vortex to mix.Transfer the 25-milliliter lysed samples into two pre-chilled S30 centrifugation tubes. Place the tubes in a centrifuge and spin at 30, 000 G for one hour at four degrees Celsius. Then, carefully recover up to 75%of the pellet-free supernatant from each S30 tube to prevent membrane contamination.Transfer the recovered supernatant into two clean precooled S30 tubes. After centrifuging the tubes again for 30 minutes, recover up to 75%of the pellet-free supernatant and transfer it into two clean 50-milliliter comical tubes. Add Ribosome Buffer A with DTT to each tube until the volume reaches 25 milliliters.Transfer the 25 milliliter samples into two clean, prechilled S100 ultracentrifuge tubes. Load the tubes into the ultracentrifuge and spin 100, 000 G for four hours at four degrees Celsius. After the final S100 centrifugation, discard the supernatant.Use a pipette to remove any residual buffer while working quickly to prevent drying of the pellet. Then add 400 microliters of ice-cold Ribosome Buffer C with DTT into each S100 tube containing the ribosomal pellet. Incubate the tubes on ice overnight in a cold room to allow the ribosome pellet to soak in the buffer.The following day, use a one-milliliter pipette and gentle swirling to fully re-suspend the clear halo ribosome pellet in buffer. Make sure only the clear halo is dissolved and avoid disturbing any yellow insoluble pellet. Then transfer the resuspended ribosome solution into two sterile two-milliliter microcentrifuge tubes.Centrifuge the tubes at 20, 000 G for 10 minutes at four degrees Celsius to remove insoluble material. Carefully transfer the pellet-free supernatant into a clean microcentrifuge tube and keep it on ice. Thaw the necessary aliquots of PURE system proteins, ribosomes, energy solution, and matrix solution on ice.Assemble a master mix for triplicate reactions by adding energy solution, PURE proteins, ribosomes, matrix solution, and nuclease-free water in the specified order. Set a pipette to 5.1 microliters and aspirate the master mix five to six times to ensure the solution is well mixed and homogeneous. Using the pipette set to 5.1 microliters, dispense the master mix into three separate wells of a 384-well optical plate, while avoiding bubbles.Seal the 384-well plate using an adhesive plate seal. All four PURE reaction batches prepared by user 1 showed consistent GFP fluorescence kinetics, peaking around two hours, and were comparable to the GFP signal from the independently prepared batch by user 2.1, while no fluorescence was observed in the negative control lacking DNA. At the two-hour time point, the GFP fluorescence for all user-prepared PURE batches was similarly high, while the minus DNA control remained near baseline.In ribosome preparations, GFP production over time varied slightly more across users with user 1.2 showing the highest activity, followed by users 2.1 and 3.1, while user 1.1 had the lowest among active batches. At 1.5 hours, user 1.2's ribosome preparation yielded the highest GFP fluorescence, while users 2.1 and 3.1 had similar but slightly lower values. User 1.1 had the lowest among active samples, and the minus DNA control remained near zero.The overall variation between users 1.1 and 1.2 is around 30%GFP production in CFPS reactions decreased progressively with increasing glycerol concentrations above 0%with 6%showing the lowest expression.

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Research

JoVE Journal

Bioengineering

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