Name: from cells to cell-free protein synthesis within 24 hours using cell-free autoinduction workflow
Uploaded: 2021-07-22T15:00:00
Description: Watch this Scientific Journal Video about From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow at JoVE.com

Authors would like to acknowledge Dr. Jennifer VanderKelen and Andrea Laubscher for technical support. Authors would also like to thank Nicole Gregorio, Max Levine, Alissa Mullin, Byungcheol So, August Brookwell, Elizabeth (Lizzy) Vojvoda, Logan Burrington and Jillian Kasman for helpful discussions. Authors also acknowledge funding support from the Bill and Linda Frost Fund, Center for Applications in Biotechnology&#39;s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and the National Science Foundation (NSF-1708919).

1. Media growth
<ol>
	<li>Prepare 960 mL of CFAI media as described in Table 1 and adjust the pH to 7.2 using KOH.</li>
	<li>Transfer culture media to a 2.5 L baffled flask and autoclave for 30 min at 121 &#176;C.</li>
	<li>Prepare a 40 mL sugar solution as described in Table 1. Filter-sterilize the solution into a separate autoclaved glass container. 
	NOTE: The sugar solution can be stored in a 30 &#176;C incubator until further use.</li>
	<li>Allow the media to completely cool ...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+synthesis+of+recombinant+proteins+from+PCR-amplified+genes+at+a+comparable+productivity+to+that+of+plasmid-based+reactions.">Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions.</a> Biochemical and Biophysical Research Communications. 338 (3), 1346-1352 (2005).</li><li>Kim, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+procedures+for+the+construction+of+a+robust+and+cost-effective+cell-free+protein+synthesis+system.">Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.</a> Journal of Biotechnology. 126 (4), 554-561 (2006).</li><li>Hunt, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streamlining+the+preparation+of+"endotoxin-free"+ClearColi+cell+extract+with+autoinduction+media+for+cell-free+protein+synthesis+of+the+therapeutic+protein+crisantaspase.">Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.</a> Synthetic and Systems Biotechnology. 4 (4), 220-224 (2019).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high-density+shaking+cultures.">Protein production by auto-induction in high-density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li><li>Shrestha, P., Holland, T. M., Bundy, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streamlined+extract+preparation+for+Escherichia+coli-based+cell-free+protein+synthesis+by+sonication+or+bead+vortex+mixing.">Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing.</a> BioTechniques. 53 (3), 163-174 (2012).</li><li>Bosdriesz, E., Molenaar, D., Teusink, B., Bruggeman, F. 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Researcher oversight is traditionally needed for two key actions during cell growth: the induction of T7 RNAP and harvesting cells at a specific OD600. CFAI obviates both of those requirements to decrease the researcher&#39;s time and technical training required in order to prepare high quality cell extracts. Auto-induction of T7 RNAP is achieved by replacing glucose with lactose as the primary sugar in the media, obviating the previous need to actively monitor the growth and then induce with IPTG&#160;at a pr...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62866">From Cells to Cell-Free Protein Synthesis within 24 Hours using Cell-Free Autoinduction Workflow</a>. The Authors&#160;section was&#160;updated.
The Authors section was updated from:
Philip E.J. Smith1,2, Taylor Slouka1,2,&#160;Javin P. Oza1,2 
1Department of Chemistry and Biochemistry, California Polytechnic State University 
2Center for Application in Biotechnology, California Polytechnic State University
to:
Philip E.J. Smith1,2, Taylor Slouka1,2, Mona Dabbas 1,2, Javin P. Oza1,2 
1Department of Chemistry and Biochemistry, California Polytechnic State University 
2Center for Application in Biotechnology, California Polytechnic State University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62866">From Cells to Cell-Free Protein Synthesis within 24 Hours using Cell-Free Autoinduction Workflow</a>. The Authors&#160;section was&#160;updated.

Erratum: From Cells to Cell-Free Protein Synthesis within 24 Hours using Cell-Free Autoinduction Workflow

The use of cell-free protein synthesis (CFPS) for biotechnology applications has grown substantially over the past few years1,2,3. This development can be attributed in part to increased efforts in understanding the processes that occur in CFPS and the role of each component4,5. Additionally, reduced costs attributed to optimized set-ups and alternative energy sources have made cell-free technology easier to implement for new users6,7,8,9.&#160;In order to implement the necessary transcription and translation factors for protein synthesis, cell extract is often used to drive cell-free reactions10. Recently published user guides have provided simple protocols for producing functional extract, making it easier to implement for new and experienced users alike1,11,12,13,14. Cell extract is usually obtained through the lysis of a cell culture, which can be grown using different organisms depending on the specific use desired1,15,16.
Escherichia coli (E. coli) has rapidly become one of the most commonly used host organisms for producing functional extracts17. The BL21 Star (DE3) strain is preferred because it removes the proteases from the outer membrane (OmpT protease) and the cytoplasm (Lon protease), providing an optimal environment for the recombinant protein expression. Additionally, the DE3 contains the &#955;DE3 that carries the gene for T7 RNA polymerase (T7 RNAP) under the control of the lacUV5 promoter; the star component contains a mutated RNaseE gene which prevents cleavage of mRNA4,14,18,19. Under the lacUV5 promoter, isopropyl-thiogalactopyranoside (IPTG) induction allows the expression of T7 RNAP20,21. These strains are used to grow and harvest cells, which give raw material for extract preparation. Cell lysis can be performed using a variety of methods, including bead beating, French press, homogenization, sonication, and nitrogen cavitation1,11,12,22.
The process of bacterial culture and harvesting is consistent across most platforms when using E. coli, but requires multiple days and intense researcher oversight1,11,13. This process generally starts with an overnight seed culture in LB broth, which upon overnight growth is then inoculated into a larger culture of 2xYTPG (yeast, tryptone, phosphate buffer, glucose) the next day. The growth of this larger culture is monitored until it reaches the early-to-mid log phase, at an optical density (OD) of 2.514,20. Constant measurement is required as the components of transcription and translation have been previously demonstrated to be highly active in the early-to-mid log phase23,24. While this process can create reproducible extract, our lab has recently developed a new method using Cell-Free Autoinduction (CFAI) Media, which reduces researcher oversight, increases the overall yield of extract for a given liter of cell culture, and improves access to E. coli-based extract preparation for both experienced and new users (Figure 1). Here we provide the step-by-step guide for implementing the CFAI workflow, to go from a streaked plate of cells to a completed CFPS reaction within 24 hours.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microfuge Tubes</td>
			<td>Phenix</td>
			<td>MPC-425Q</td>
			<td></td>
		</tr>
		<tr>
			<td>1L Centrifuge Tube</td>
			<td>Beckman Coulter</td>
			<td>A99028</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-E Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369001</td>
			<td></td>
		</tr>
		<tr>
			<td>CoA</td>
			<td>Sigma-Aldrich</td>
			<td>C3144-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td>Biotek</td>
			<td>BTCYT5F</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher</td>
			<td>D16-3</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Lactose</td>
			<td>Alfa Aesar</td>
			<td>J66376</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>ThermoFisher</td>
			<td>15508013</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F7878-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher</td>
			<td>11344041</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>JLA-8.1000 Rotor</td>
			<td>Beckman Coulter</td>
			<td>366754</td>
			<td></td>
		</tr>
		<tr>
			<td>K(Glu)</td>
			<td>Sigma-Aldrich</td>
			<td>G1501-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>K(OAc)</td>
			<td>Sigma-Aldrich</td>
			<td>P1190-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>P5958-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Sigma-Aldrich</td>
			<td>A7627-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A8094-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Sigma-Aldrich</td>
			<td>A0884-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A7219-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G1501-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histadine</td>
			<td>Sigma-Aldrich</td>
			<td>H8000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I2752-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M9625-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P2126-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Sigma-Aldrich</td>
			<td>T8625-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophan</td>
			<td>Sigma-Aldrich</td>
			<td>T0254-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T3754-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth</td>
			<td>ThermoFisher</td>
			<td>12795027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma-Aldrich</td>
			<td>V0500-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Glu)2</td>
			<td>Sigma-Aldrich</td>
			<td>49605-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(OAc)2</td>
			<td>Sigma-Aldrich</td>
			<td>M5661-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 20</td>
			<td>Beckman Coulter</td>
			<td>B30134</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Grade Water</td>
			<td>Sigma-Aldrich</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Alfa Aesar</td>
			<td>A12313</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>Sigma-Aldrich</td>
			<td>N8535-15VL</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 42/42R Incubator</td>
			<td>Eppendorf</td>
			<td>M1335-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4(Glu)</td>
			<td>Sigma-Aldrich</td>
			<td>09689-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NTPs</td>
			<td>ThermoFisher</td>
			<td>R0481</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxalic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>P0963-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEP</td>
			<td>Sigma-Aldrich</td>
			<td>860077-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Acros, Organics</td>
			<td>A0382124</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Acros, Organics</td>
			<td>A0379904</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink HiPure Plasmid Prep Kit</td>
			<td>ThermoFisher</td>
			<td>K210007</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>D13208-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>S0266-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(OAc)</td>
			<td>Sigma-Aldrich</td>
			<td>T6066-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma-Aldrich</td>
			<td>10109541001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>73049-73-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Tunair 2.5L Baffled Shake Flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z710822</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Processor</td>
			<td>QSonica</td>
			<td>Q125-230V/50HZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Bioreagents</td>
			<td>1/2/8013</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

When preparing CFAI media, glucose was exchanged for an increase in lactose and glycerol as the main energy substrate in the media. Additionally, the buffering capacity of the CFAI media was increased as well. These specific components are given in Table 1.
The cells were then grown to both an OD600 of 10 and the standard 2.5 in CFAI media to show consistency with extract quality despite varying extract quantities. The 2.5 OD600 CFAI media was grown after...

Watch this Scientific Journal Video about From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow at JoVE.com

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

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 ...

The cell-free autoinduction method reduces researcher oversight and time needed to produce functional cell extracts. This simplifies previous in vitro protein synthesis protocols that were more time-consuming and intensive. This technique is less complicated and also high yielding, which will enable a larger audience to implement cell-free expression.This method highlights the importance of energy metabolism during cell growth and within cell-free reactions. Begin by preparing 960 milliliters of cell-free autoinduction media. Then, sterilize the media in a 2.5-liter baffled flask by autoclaving for 30 minutes at 121 degrees Celsius.Next, after preparing 40 milliliters of the sugar solution, filter-sterilize it into a separate autoclaved glass container. After autoclaving, when the culture media has cooled down to below 40 degrees Celsius, add the filter-sterilized sugar solution directly to the media. Next, inoculate the media by swiping a loop full of colonies from a previously streaked fresh E.coli BL21 star plate and inserting the loop directly into the media.Swirl the loop into the media without touching the sides of the container. Then, place the inoculated media overnight in a 30 degree Celsius incubator with shaking at 200 RPM. The next day, harvest the cells by transferring one liter of the media into a one-liter centrifuge bottle and centrifuging at 5, 000 times G between four to 10 degrees Celsius for 10 minutes.After discarding the supernatant, use a sterile spatula to transfer the pellet to a pre-chilled and previously weighed 50-milliliter conical tube. Next, wash the pellet once with 30 to 40 milliliters of cold S30 buffer by resuspending the pellet via vortexing in 30-second bursts with rest periods on ice. After the pellet is completely resuspended, centrifuge the cell resuspension, discard the supernatant, and using a clean tissue, wipe any excess supernatant from the inside walls of the 50-milliliter tube without touching the pellet.Then, weigh the pellet before flash freezing it in liquid nitrogen and store it at minus 80 degrees Celsius until further use. To prepare the cell extract, add one milliliter of S30 buffer per gram of the frozen cell pellet and allow the pellet to thaw on ice for 30 to 60 minutes. Then, resuspend the thawed pellet via vortexing in bursts of 30 seconds with rest periods on ice until no visible cell clumps remain.For cell lysis, transfer 1.4 milliliter aliquots of the cell suspension into 1.5-milliliter microfuge tubes. Then, sonicate the cell suspension in each tube with a frequency of 20 kilohertz and 50%amplitude for three bursts of 45 seconds with 59 seconds of rest per cycle in an ice bath Invert the tube between cycles, and after the last sonication cycle, immediately add 4.5 microliters of one-molar dithiothreitol. After sonicating the cell suspension in all tubes, centrifuge the tubes and collect the supernatant in 600-microliter aliquots into fresh 1.5-milliliter microfuge tubes.Flash freeze and store the aliquots at minus 80 degrees Celsius until further use. To perform 15 microliters of cell-free protein synthesis reaction in 1.5-milliliter microfuge tubes in quadruplicate, thaw one aliquot of the cell extract. After preparing each 15-microliter reaction mixture, allow the reaction to run for at least four hours at 37 degrees Celsius.To quantify the reporter protein, combine 48 microliters of 50-millimolar HEPES buffer at pH 7.2 with two microliters of each cell-free protein synthesis reaction product in a black polystyrene 96-well plate. Quantify the fluorescence intensity of the superfolder green fluorescent protein, or sfGFP, using an excitation wavelength of 485 and emission wavelength of 528 nanometers. Then, convert the relative fluorescence units to the volumetric yield of sfGFP by creating a standard curve using purified pJL1 sfGFP plasmid.Shown here are cell-free autoinduction media pellets after cell harvest at different optical densities measured at 600 nanometers. The media grown to an optical density of 10 produced a higher amount of cell pellet in overall extract than the media grown to an optical density of 2.5. Further analysis of total protein concentration demonstrated no significant difference in overall protein between both extracts.Even though the media was grown to different optical density levels, both extracts demonstrated similar results in cell-free reactions expressing sfGFP. Maintaining sterile conditions while preparing the media and sugar solution is important. This CFAI cell-extract method can be used for most applications of cell-free expression.These range from biotechnology education to protein engineering, as well as point-of-care diagnostics. This method reduces the amount of time and technical skill required, improves reproducibility, and produces higher quantities of extract.

Research

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Bioengineering

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

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Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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

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