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>
	<li>Gregorio, N. E., Levine, M. Z., Oza, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+cell-free+protein+synthesis.">A user's guide to cell-free protein synthesis.</a> Methods and Protocols. 2 (1), 1-34 (2019).</li><li>Silverman, A. D., Karim, A. S., Jewett, M. C. <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+gene+expression:+an+expanded+repertoire+of+applications.">Cell-free gene expression: an expanded repertoire of applications.</a> Nature Reviews Genetics. 21 (3), (2020).</li><li>Swartz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+biological+applications+using+cell-free+metabolic+engineering:+An+overview.">Expanding biological applications using cell-free metabolic engineering: An overview.</a> Metabolic Engineering. 50, 156-172 (2018).</li><li>Jared, B., Dopp, L., Tamiev, D. D., Reuel, N. F. <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+supplement+mixtures:+Elucidating+the+history+and+biochemical+utility+of+additives+used+to+support+in+vitro+protein+synthesis+in+E.+coli+extract.">Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract.</a> Biotechnology Advances. 37 (1), 246-258 (2019).</li><li>Jewett, M. C., Swartz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+the+Escherichia+coli+cytoplasmic+environment+activates+long-lived+and+efficient+cell-free+protein+synthesis.">Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis.</a> Biotechnology and Bioengineering. 86 (1), 19-26 (2004).</li><li>Calhoun, K. A., Swartz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energizing+cell-free+protein+synthesis+with+glucose+metabolism.">Energizing cell-free protein synthesis with glucose metabolism.</a> Biotechnology and Bioengineering. 90 (5), 606-613 (2005).</li><li>Levine, M. Z., Gregorio, N. E., Jewett, M. C., Watts, K. R., Oza, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli-based+cell-free+protein+synthesis:+Protocols+for+a+robust,+flexible,+and+accessible+platform+technology.">Escherichia coli-based cell-free protein synthesis: Protocols for a robust, flexible, and accessible platform technology.</a> Journal of Visualized Experiments. (144), e58882 (2019).</li><li>Sun, Z. Z., 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=Protocols+for+Implementing+an+Escherichia+coli+Based+TX-TL+Cell-Free+Expression.">Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression.</a> System for Synthetic Biology. , 1-14 (2013).</li><li>Pardee, K., 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=Portable,+on-demand+biomolecular+manufacturing.">Portable, on-demand biomolecular manufacturing.</a> Cell. 167, 248-254 (2016).</li><li>Moore, S. J., Macdonald, J. T., Freemont, P. S. <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+synthetic+biology+for+in+vitro+prototype+engineering.">Cell-free synthetic biology for in vitro prototype engineering.</a> Biochemical Society Transactions. 45 (3), 785-791 (2017).</li><li>Cole, S. D., Miklos, A. E., Chiao, A. C., Sun, Z. Z., Lux, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+preparation+of+prokaryotic+extracts+for+cell-free+expression+systems.">Methodologies for preparation of prokaryotic extracts for cell-free expression systems.</a> Synthetic and Systems Biotechnology. 5 (4), 252-267 (2020).</li><li>Didovyk, A., Tonooka, T., Tsimring, L., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+scalable+preparation+of+bacterial+lysates+for+cell-free+gene+expression.">Rapid and scalable preparation of bacterial lysates for cell-free gene expression.</a> ACS Synthetic Biology. 6 (12), 2198-2208 (2017).</li><li>Dopp, J. L., Reuel, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+optimization+for+scalable+E.+coli+extract+preparation+for+cell-free+protein+synthesis.">Process optimization for scalable E. coli extract preparation for cell-free protein synthesis.</a> Biochemical Engineering Journal. 138, 21-28 (2018).</li><li>Kwon, Y. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+preparation+methods+of+crude+extract+for+robust+cell-free+protein+synthesis.">High-throughput preparation methods of crude extract for robust cell-free protein synthesis.</a> Scientific Reports. 5, 8663 (2015).</li><li>Endo, Y., Sawasaki, T. <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+expression+systems+for+eukaryotic+protein+production.">Cell-free expression systems for eukaryotic protein production.</a> Current Opinion in Biotechnology. 17 (4), 373-380 (2006).</li><li>Zemella, A., Thoring, L., Hoffmeister, C., Kubick, S. <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+protein+synthesis:+Pros+and+cons+of+prokaryotic+and+eukaryotic+systems.">Cell-free protein synthesis: Pros and cons of prokaryotic and eukaryotic systems.</a> ChemBioChem. 16 (17), 2420-2431 (2015).</li><li>Laohakunakorn, N., Grasemann, L., Lavickova, B., Michielin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-up+construction+of+complex+biomolecular+systems+with+cell-free+synthetic+biology.">Bottom-up construction of complex biomolecular systems with cell-free synthetic biology.</a> Frontiers in Bioengineering and Biotechnology. 8, (2020).</li><li>Ahn, J. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+fast-growing+bacteria+robustly+tune+their+ribosome+concentration+to+approximate+growth-rate+maximization.">How fast-growing bacteria robustly tune their ribosome concentration to approximate growth-rate maximization.</a> FEBS Journal. 282 (10), 2029-2044 (2015).</li><li>Zawada, J., Swartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+rapid+growth+in+moderate-density+Escherichia+coli+fermentations.">Maintaining rapid growth in moderate-density Escherichia coli fermentations.</a> Biotechnology and Bioengineering. 89 (4), 407-415 (2005).</li><li>Kim, J., Copeland, C. E., Padumane, S. R., Kwon, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crude+extract+preparation+and+optimization+from+a+genomically+engineered+Escherichia+coli+for+the+cell-free+protein+synthesis+system:+Practical+laboratory+guideline.">A crude extract preparation and optimization from a genomically engineered Escherichia coli for the cell-free protein synthesis system: Practical laboratory guideline.</a> Methods and Protocols. 2 (3), 1-15 (2019).</li><li>Failmezger, J., Rauter, M., Nitschel, R., Kraml, M., Siemann-Herzberg, M. <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+protein+synthesis+from+non-growing,+stressed+Escherichia+coli.">Cell-free protein synthesis from non-growing, stressed Escherichia coli.</a> Scientific Reports. 7 (1), 1-10 (2017).</li><li>Levine, M. Z., 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=Activation+of+energy+metabolism+through+growth+media+reformulation+enables+a+24-h+workflow+for+cell-free+expression.">Activation of energy metabolism through growth media reformulation enables a 24-h workflow for cell-free expression.</a> ACS Synthetic Biology. 9 (10), 2765-2774 (2020).</li><li>Martin, R. 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=Cell-free+protein+synthesis+from+genomically+recoded+bacteria+enables+multisite+incorporation+of+noncanonical+amino+acids.">Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.</a> Nature Communications. , 1-9 (2018).</li><li>Soye, B. J. D., 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=Resource+A+highly+productive,+One-pot+cell-free+protein+synthesis+platform+based+on+genomically+recoded+Escherichia+coli+resource.">Resource A highly productive, One-pot cell-free protein synthesis platform based on genomically recoded Escherichia coli resource.</a> Cell Chemical Biology. 26 (12), 1743-1754 (2019).</li><li>Ezure, T., Suzuki, T., Ando, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+protein+synthesis+system+from+insect+cells.">A cell-free protein synthesis system from insect cells.</a> Methods in Molecular Biology. , 285-296 (2014).</li><li>Heide, C., 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=development+and+optimization+of+a+functional+mammalian+cell-free+protein+synthesis+platform.">development and optimization of a functional mammalian cell-free protein synthesis platform.</a> Frontiers in Bioengineering and Biotechnology. 8, 1-10 (2021).</li></ol>

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...

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 ...

Research

JoVE Journal

Bioengineering

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

科研

生物工程

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

使用蛋白质膜吸收器和流线型工作流的奇美人 Fc 表达蛋白质的隔离和特征

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

密封飞升商会阵列的无细胞生物学

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

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

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

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

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

快速生长的纳氏弧菌的无细胞蛋白表达

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

用于长期无细胞基因表达传导的多层微流体平台

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

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

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

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

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

OnePot PURE Cell-Free System

一锅纯无细胞系统

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...