Name: preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer
Uploaded: 2020-04-27T13:00:00
Description: Watch this Scientific Journal Video about Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer at JoVE.com

This work was supported by ERC-STG-2015-680242.
The authors also acknowledge the support of the Technion Integrated Cancer Center (TICC); the Russell Berrie Nanotechnology Institute; the Lorry I. Lokey Interdisciplinary Center for Life Sciences &#38; Engineering; the Israel Ministry of Economy for a Kamin Grant (52752); the Israel Ministry of Science Technology and Space &#8211; Office of the Chief Scientist (3-11878); the Israel Science Foundation (1778/13, 1421/17); the Israel Cancer Association (2015-0116); the German-Israeli Foundation for Scientific Research and Development for a GIF Young grant (I-2328-1139.10/2012); the European Union FP-7 IRG Program for a Career Integration Grant (908049); the Phospholipid Research Center Grant; a Rosenblatt Foundation for cancer research,&#160;a Mallat Family Foundation Grant; and the Unger Family Foundation. A. Schroeder acknowledges Alon and Taub Fellowships. O. Adir acknowledges the Sherman and Gutwirth fellowships. G. Chen acknowledges the Sherman Fellowship. N. Krinsky acknowledges the Baroness Ariane de Rothschild Women Doctoral Program from the Rothschild Caesarea Foundation.

NOTE: Illustration of the complete synthetic cells&#8217; production protocol is presented in Figure 1. According to the user&#8217;s needs, the protein expression (section 3.2) and synthetic cell formation (section 4) parts of the protocol can also be carried out independently (with some adaptations).
1. Preparation of S30-T7 lysate
<ol>
	<li>Streak plate the E. coli BL21(DE3) bacteria transformed with the T7 RNA polymerase expressing pAR1219 plasmid o...

<ol>
	<li>Blain, J. C., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+synthetic+cells.">Progress toward synthetic cells.</a> Annual review of biochemistry. 83, 615-640 (2014).</li><li>Richmond, D. L., 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=Forming+giant+vesicles+with+controlled+membrane+composition,+asymmetry,+and+contents.">Forming giant vesicles with controlled membrane composition, asymmetry, and contents.</a> Proceedings of the National Academy of Sciences. 108 (23), 9431-9436 (2011).</li><li>Deshpande, S., Spoelstra, W. K., van Doorn, M., Kerssemakers, J., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+division+of+cell-sized+liposomes.">Mechanical division of cell-sized liposomes.</a> ACS Nano. 12 (3), 2560-2568 (2018).</li><li>Liu, D. V., Zawada, J. F., 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=Streamlining+Escherichia+coli+S30+extract+preparation+for+economical+cell-free+protein+synthesis.">Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis.</a> Biotechnology Progress. 21 (2), 460-465 (2005).</li><li>Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+Inside+Liposomes:+From+Early+Studies+to+Current+Protocols.">Gene Expression Inside Liposomes: From Early Studies to Current Protocols.</a> Chemistry-A European Journal. , (2019).</li><li>Lewandowski, B., 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=Sequence-specific+peptide+synthesis+by+an+artificial+small-molecule+machine.">Sequence-specific peptide synthesis by an artificial small-molecule machine.</a> Science. 339 (6116), 189-193 (2013).</li><li>Li, J., 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=Cogenerating+synthetic+parts+toward+a+self-replicating+system.">Cogenerating synthetic parts toward a self-replicating system.</a> ACS Synthetic Biology. 6 (7), 1327-1336 (2017).</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=An+economical+and+highly+productive+cell-free+protein+synthesis+system+utilizing+fructose-1,6-bisphosphate+as+an+energy+source.">An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source.</a> Journal of Biotechnology. 130 (4), 389-393 (2007).</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>Noireaux, V., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vesicle+bioreactor+as+a+step+toward+an+artificial+cell+assembly.">A vesicle bioreactor as a step toward an artificial cell assembly.</a> Proceedings of the National Academy of Sciences. 101 (51), 17669-17674 (2004).</li><li>He, M., He, Y., Luo, Q., Wang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+DNA+to+protein:+No+living+cells+required.">From DNA to protein: No living cells required.</a> Process Biochemistry. 46 (3), 615-620 (2011).</li><li>Casteleijn, M. G., Urtti, A., Sarkhel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+without+boundaries:+cell-free+protein+synthesis+in+pharmaceutical+research.">Expression without boundaries: cell-free protein synthesis in pharmaceutical research.</a> International Journal of Pharmaceutics. 440 (1), 39-47 (2013).</li><li>Luisi, P. L., Ferri, F., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+semi-synthetic+minimal+cells:+a+review.">Approaches to semi-synthetic minimal cells: a review.</a> Naturwissenschaften. 93 (1), 1-13 (2006).</li><li>Adamala, K. P., Martin-Alarcon, D. A., Guthrie-Honea, K. R., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+genetic+circuit+interactions+within+and+between+synthetic+minimal+cells.">Engineering genetic circuit interactions within and between synthetic minimal cells.</a> Nature Chemistry. 9 (5), 431-439 (2017).</li><li>Ding, Y., Contreras-Llano, L. E., Morris, E., Mao, M., Tan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+Context+Dependency+of+Gene+Networks+Using+Artificial+Cells.">Minimizing Context Dependency of Gene Networks Using Artificial Cells.</a> ACS Applied Materials &amp; Interfaces. 10 (36), 30137-30146 (2018).</li><li>Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+by+directed+evolution.">Design by directed evolution.</a> Accounts of Chemical Research. 31 (3), 125-131 (1998).</li><li>Chen, 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=Synthetic+beta+cells+for+fusion-mediated+dynamic+insulin+secretion.">Synthetic beta cells for fusion-mediated dynamic insulin secretion.</a> Nature Chemical Biology. 14 (1), 86-93 (2018).</li><li>Mohr, B. P., Retterer, S. T., Doktycz, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=While-you-wait+proteins?+Producing+biomolecules+at+the+point+of+need.">While-you-wait proteins? Producing biomolecules at the point of need.</a> Expert Review of Proteomics. 13 (8), 707-709 (2016).</li><li>Krinsky, N., 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=Synthetic+Cells+Synthesize+Therapeutic+Proteins+inside+Tumors.">Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.</a> Advanced Healthcare Materials. 7 (9), 1701163 (2018).</li><li>Kim, T. W., Kim, D. M., Choi, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+production+of+milligram+quantities+of+proteins+in+a+batch+cell-free+protein+synthesis+system.">Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system.</a> Journal of Biotechnology. 124 (2), 373-380 (2006).</li><li>Deng, N. N., Yelleswarapu, M., Zheng, L., Huck, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+assembly+of+monodisperse+vesosomes+as+artificial+cell+models.">Microfluidic assembly of monodisperse vesosomes as artificial cell models.</a> Journal of the American Chemical Society. 139 (2), 587-590 (2016).</li><li>Deshpande, S., Caspi, Y., Meijering, A. E., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octanol-assisted+liposome+assembly+on+chip.">Octanol-assisted liposome assembly on chip.</a> Nature Communications. 7, 10447 (2016).</li><li>Go&#776;pfrich, 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=One-Pot+Assembly+of+Complex+Giant+Unilamellar+Vesicle-Based+Synthetic+Cells.">One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells.</a> ACS synthetic biology. , (2019).</li><li>Fujii, S., 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=Liposome+display+for+in+vitro+selection+and+evolution+of+membrane+proteins.">Liposome display for in vitro selection and evolution of membrane proteins.</a> Nature Protocols. 9 (7), 1578 (2014).</li><li>Periasamy, A., 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+of+membrane+(1,3)-beta-d-glucan+(curdlan)+synthase:+co-translational+insertion+in+liposomes+and+reconstitution+in+nanodiscs.">Cell-free protein synthesis of membrane (1,3)-beta-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.</a> Biochim Biophys Acta. 1828 (2), 743-757 (2013).</li><li>Kalmbach, R., 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=Functional+cell-free+synthesis+of+a+seven+helix+membrane+protein:+in+situ+insertion+of+bacteriorhodopsin+into+liposomes.">Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes.</a> Journal of Molecular Biology. 371 (3), 639-648 (2007).</li><li>Nishimura, 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=Cell-free+protein+synthesis+inside+giant+unilamellar+vesicles+analyzed+by+flow+cytometry.">Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry.</a> Langmuir. 28 (22), 8426-8432 (2012).</li><li>Rampioni, G., 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=Synthetic+cells+produce+a+quorum+sensing+chemical+signal+perceived+by+Pseudomonas+aeruginosa.">Synthetic cells produce a quorum sensing chemical signal perceived by Pseudomonas aeruginosa.</a> Chemical Communications. 54 (17), 2090-2093 (2018).</li><li>Stano, P., Kuruma, Y., de Souza, T. P., Luisi, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosynthesis of proteins inside liposomes. , 127-145 (2010).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+unilamellar+vesicles+using+an+inverted+emulsion.">Production of unilamellar vesicles using an inverted emulsion.</a> Langmuir. 19 (7), 2870-2879 (2003).</li><li>Krinsky, N., 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=A+Simple+and+Rapid+Method+for+Preparing+a+Cell-Free+Bacterial+Lysate+for+Protein+Synthesis.">A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.</a> PLoS One. 11 (10), 0165137 (2016).</li><li>P&#233;delacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., Waldo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+a+superfolder+green+fluorescent+protein.">Engineering and characterization of a superfolder green fluorescent protein.</a> Nature Biotechnology. 24 (1), 79 (2006).</li><li>Osawa, M., Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+division+by+a+simple+bacterial+division+machinery.">Liposome division by a simple bacterial division machinery.</a> Proceedings of the National Academy of Sciences. 110 (27), 11000-11004 (2013).</li><li>Merkle, D., Kahya, N., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+and+anchoring+of+cytoskeleton+inside+giant+unilamellar+vesicles.">Reconstitution and anchoring of cytoskeleton inside giant unilamellar vesicles.</a> ChemBioChem. 9 (16), 2673-2681 (2008).</li><li>Vleugel, M., Roth, S., Groenendijk, C. F., Dogterom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+basic+mitotic+spindles+in+spherical+emulsion+droplets.">Reconstitution of basic mitotic spindles in spherical emulsion droplets.</a> Journal of Visualized Experiments. , e54278 (2016).</li><li>Bayoumi, M., Bayley, H., Maglia, G., Sapra, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-compartment+encapsulation+of+communicating+droplets+and+droplet+networks+in+hydrogel+as+a+model+for+artificial+cells.">Multi-compartment encapsulation of communicating droplets and droplet networks in hydrogel as a model for artificial cells.</a> Scientific Reports. 7, 45167 (2017).</li><li>Carlson, E. D., Gan, R., Hodgman, C. E., 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+protein+synthesis:+applications+come+of+age.">Cell-free protein synthesis: applications come of age.</a> Biotechnology Advances. 30 (5), 1185-1194 (2012).</li></ol>

This protocol introduces a simple and affordable method for the production of large quantities of protein-producing synthetic cells. The yield of active cells is dependent on careful and accurate execution of the protocol with emphasis on several critical steps. In the lysate preparation section of this method, it is essential to reach the appropriate bacteria density before cell lysis to achieve a sufficient amount of proteins in the bacterial lysate. Second, the lysis process should be performed at 4 &#176;C and the ly...

Synthetic cells are artificial cell-like particles, mimicking one or multiple functions of a living cell, such as the ability to divide, form membrane interactions, and synthesize proteins based on a genetic code1,2,3. Synthetic cells that enclose cell-free protein synthesis (CFPS) systems possess high modularity due to their ability to produce various proteins and RNA sequences following alterations in the DNA template. Presenting an attractive alternative to the current approaches of protein production, CFPS systems are based on cell lysate, purified components, or synthetic components and include all the transcription and translation machinery required for protein synthesis such as ribosomes, RNA polymerase, amino acids and energy sources (e.g., 3-phosphoglycerate and adenine triphosphate)4,5,6,7,8,9. The encapsulation of a CFPS system inside lipid vesicles enables the simple and efficient production of proteins without depending on a living cell10. Moreover, this platform allows synthesis of peptides that may degrade inside natural cells, production of&#160;proteins that are toxic to living cells, and modify proteins with non-natural amino acids11,12. Synthetic cells have been used as a model for research purposes investigating the minimal cell components required to enable cellular life from an evolutionary perspective1,13. Synthetic cells have also been used to build and implement genetic circuit and as models for directed evolution14,15,16. Other studies have focused on the ability of synthetic cells to mimic the biological activity of natural cells, aiming to replace damaged natural cells, such as beta cells in patients with diabetes17. Furthermore, the ability of these CFPS encapsulating synthetic cells to produce a variety of therapeutic proteins illustrates its potential to be incorporated into clinical use18.
Here we describe a bottom-up lab-scale protocol (Figure 1) for the production of RNA and protein-producing synthetic cells based on a CFPS system encapsulated in a lipid vesicle. This shows the potential use of synthetic cell platforms as novel drug delivery systems for the onsite production of a therapeutic protein drug in vivo19. Previous studies have investigated the optimization of the CFPS reaction and the cell lysate preparation processes4,8,20. Moreover, several techniques have been applied for cell-sized liposome preparation, such as microfluidic and polymer-based droplet stabilization methods21,22,23, which also differ in the liposomes&#39; lipid composition24,25,26. In the presented protocol, synthetic cells are produced using a water-in-oil emulsion transfer method and the encapsulation process is carried out at low temperatures (&#60;4 &#176;C)5,10,24,27,28. These mild conditions have been found to be favorable for retaining the bio-functional integrity of the molecular machinery, namely ribosomes and proteins27,29,30. The lipid composition of the particles consists of both cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The first is found in all mammalian cell membranes and is essential for the stability, rigidity and permeability reduction of the membrane, and the latter mimics mammalian phospholipid composition11,13. The cellular transcription and translation molecular machinery are extracted from the BL21 (DE3) Escherichia coli (E. coli) strain, which is transformed with pAR1219 plasmid overexpressing T7 RNA polymerase to increase CFPS potency and protein synthesis. This system has been used to produce diagnostic and therapeutic proteins, with molecular weights of up to 66 kDa in vitro and in vivo19,31. The following protocol provides a simple and effective method for the production of the synthetic cell system, which can address a wide range of fundamental questions associated with protein synthesis in nature and can also be utilized for drug delivery applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td colspan="4" >A. Reagents required for step 1 (S30-T7 lysate preparation)</td>
		</tr>
		<tr >
			<td >E.coli BL21 (DE3)&#160;</td>
			<td >NEB</td>
			<td >C2527</td>
			<td >E.coli BL21 (DE3).</td>
		</tr>
		<tr >
			<td >pAR1219</td>
			<td >Sigma</td>
			<td >T2076</td>
			<td >TargeTron vector for transformation.</td>
		</tr>
		<tr >
			<td >Stock solution of 50 mg/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
			<td >Stored at -20 &#176;C.</td>
		</tr>
		<tr >
			<td >10 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="5" >For preparation of Luria Bertani (LB) agar (1.5%) plate.</td>
		</tr>
		<tr >
			<td >10 g/L Sodium chloride (NaCl)</td>
			<td >Bio-Lab</td>
			<td>19030591</td>
		</tr>
		<tr >
			<td >5 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >15 g/L Agar agar purified</td>
			<td >Merck</td>
			<td>1.01614.5007</td>
		</tr>
		<tr >
			<td >50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >10 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="4" >For preparation of Luria Bertani (LB) media (20 mL).</td>
		</tr>
		<tr >
			<td >10 g/L Sodium chloride (NaCl)</td>
			<td >Bio-Lab</td>
			<td>19030591</td>
		</tr>
		<tr >
			<td >5 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >12 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="6" >For preparation of Terrific Broth (TB) media (1 L).</td>
		</tr>
		<tr >
			<td >24 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >4% (v/v) Glycerol anhydrous</td>
			<td >Bio-Lab</td>
			<td >7120501</td>
		</tr>
		<tr >
			<td >2.32 g/L K2HPO4</td>
			<td>Spectrum chemical</td>
			<td>P1383</td>
		</tr>
		<tr >
			<td >12.54 g/L KH2PO4</td>
			<td>Spectrum chemical</td>
			<td>P1380</td>
		</tr>
		<tr >
			<td >&#160;50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >Stock solution of 100 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td >INALCO</td>
			<td >INA-1758-1400</td>
			<td >Filtered using 0.2 &#181;m hydrophilic PVDF syringe filter.</td>
		</tr>
		<tr >
			<td >Stock solution of 0.1 M dithiothreitol (DTT)&#160;</td>
			<td >TCI</td>
			<td >D1071</td>
			<td >Filtered using 0.2 &#181;m hydrophilic PVDF syringe filter.</td>
		</tr>
		<tr >
			<td >10 mM Tris-acetate at pH = 7.4</td>
			<td >Sigma</td>
			<td >T1503</td>
			<td rowspan="5" >S30 lysate buffer (1.5 L)</td>
		</tr>
		<tr >
			<td >14 mM magnesium acetate</td>
			<td >Merck</td>
			<td >1.05819.0250</td>
		</tr>
		<tr >
			<td >60 mM potassium acetate</td>
			<td >Carlo Erba</td>
			<td >470147</td>
		</tr>
		<tr >
			<td >1 mM DTT</td>
			<td >TCI</td>
			<td >D1071</td>
		</tr>
		<tr >
			<td >0.5 mL/L 2-mercaptoethanol</td>
			<td >Sigma</td>
			<td >M6250</td>
		</tr>
		<tr >
			<td colspan="4" >Equipment required for step 1</td>
		</tr>
		<tr >
			<td >100 mL sterilized Erlenmeyer flasks</td>
			<td >Thermo Scientific</td>
			<td >50-154-2846</td>
			<td >2 flasks</td>
		</tr>
		<tr >
			<td >2 L sterilized Erlenmeyer flasks with baffles</td>
			<td >KIMAX-KIMBLE</td>
			<td >25630</td>
			<td >2 flasks</td>
		</tr>
		<tr >
			<td >Floor incubator shaker</td>
			<td >MRC</td>
			<td >TOU-120-2</td>
			<td >Laboratory shaker incubator 450x450mm, 400rpm, 70 &#176;C</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75004270</td>
			<td >(75003340) - Fiberlite F10-6 x 100 LEX Fixed-Angle Rotor. 
			Should enable at least 13,000 x g.&#160;&#160;&#160;&#160; * Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >High pressure homogenizer</td>
			<td >AVESTIN</td>
			<td>EmulsiFlex-C3</td>
			<td >Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >-80oC freezer</td>
			<td>SO-LOW</td>
			<td>U85-18</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized 1.5 mL plastic tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Spectrophotometer</td>
			<td >TECAN</td>
			<td >IN-MNANO</td>
			<td >Infinite M200 pro</td>
		</tr>
		<tr >
			<td >96-well transparent plate</td>
			<td >Thermo Scientific</td>
			<td>167008</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized graduated cylinder</td>
			<td >Corning</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Sterilized pipette tips</td>
			<td >Corning</td>
			<td ></td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Crushed ice bucket</td>
			<td>Bel-Art</td>
			<td >M18848-4001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Small liquid nitrogen tank</td>
			<td>NALGENE</td>
			<td>4150-4000</td>
			<td ></td>
		</tr>
		<tr >
			<td colspan="4" >B. Reagents required for step 3 (lipids in oil solution preparation):</td>
		</tr>
		<tr >
			<td >1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td>
			<td >Lipoid</td>
			<td >556400</td>
			<td >Powder</td>
		</tr>
		<tr >
			<td >Cholesterol</td>
			<td >Sigma</td>
			<td >C8667</td>
			<td >Powder</td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >Bio-Lab</td>
			<td >3082301</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >Sigma</td>
			<td >M5904</td>
			<td >Light oil</td>
		</tr>
		<tr >
			<td colspan="4" >Equipment required for step 2</td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td >Scientific industries</td>
			<td >SI-0256</td>
			<td ></td>
		</tr>
		<tr >
			<td >Heating block</td>
			<td >TECHNE</td>
			<td >FDB03AD</td>
			<td >Pre-heated to 80 &#176;C. 
			Should enable controlled temperature.&#160;</td>
		</tr>
		<tr >
			<td >2 mL screw neck glass vials</td>
			<td >CSI Analytical Innovations</td>
			<td >VT009M-1232</td>
			<td rowspan="2" >For a larger scale, use 50 mL falcons and evaporate the chloroform using rotary evaporator.</td>
		</tr>
		<tr >
			<td >9mm Screw Cap</td>
			<td >CSI Analytical Innovations</td>
			<td >C395R-09LC</td>
		</tr>
		<tr >
			<td colspan="4" >C. Reagents required for step 3 (inner and feeding reaction mixtures):</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >Spectrum</td>
			<td >H1089</td>
			<td rowspan="2" >1 M HEPES-KOH (pH = 8) - pH buffer</td>
		</tr>
		<tr >
			<td >Potassium hydroxide (KOH)</td>
			<td >Frutarom</td>
			<td >55290</td>
		</tr>
		<tr >
			<td >1 M Magnesium acetate</td>
			<td >Merck</td>
			<td >1.05819.0250</td>
			<td >Co-factor and negative charge stabilizor.</td>
		</tr>
		<tr >
			<td >1 M Potassium acetate</td>
			<td >Carlo Erba</td>
			<td >470147</td>
			<td >Negative charge stabilizor.</td>
		</tr>
		<tr >
			<td >5.2 M Ammonium acetate</td>
			<td >Merck</td>
			<td >1.01116.1000</td>
			<td >Stabilizes negative charge.</td>
		</tr>
		<tr >
			<td >50% (w/v) Polyethylene glycol 6000 (PEG)</td>
			<td >Merck</td>
			<td >8.07491.1000</td>
			<td >Increases the concentration of the macromolecules.</td>
		</tr>
		<tr >
			<td >0.5 M 3-phosphoglycerate (3-PGA)</td>
			<td>Sigma</td>
			<td>P8877</td>
			<td >Secondary energy source.</td>
		</tr>
		<tr >
			<td >50 mM Amino acids mixture I</td>
			<td>Sigma</td>
			<td>LAA21-1KT</td>
			<td >Amino acids additive. 
			Contains: 50 mM of each of the following 17&#160; natural amino acids - alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine.</td>
		</tr>
		<tr >
			<td >50 mM Amino acids mixture II</td>
			<td>Sigma</td>
			<td>LAA21-1KT</td>
			<td >Amino acids additive. 
			Contains: 50 mM of each of the following 3 natural amino acids - tryptophan, phenylalanine, and tyrosine.</td>
		</tr>
		<tr >
			<td >100 mM Adenine triphosphate (ATP)</td>
			<td>Sigma</td>
			<td>A3377</td>
			<td >Nucleotides &#38; energy source.</td>
		</tr>
		<tr >
			<td >50 mM Guanidine triphosphate (GTP)</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td >Nucleotides &#38; energy source.</td>
		</tr>
		<tr >
			<td >100 mM Uridine triphosphate (UTP)</td>
			<td>ACROS ORGANICS</td>
			<td>226310010</td>
			<td >Nucleotides additive.</td>
		</tr>
		<tr >
			<td >100 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td >INALCO</td>
			<td >INA-1758-1400</td>
			<td >Genes expression induction.</td>
		</tr>
		<tr >
			<td >2 M Sucrose</td>
			<td >J.T. Baker</td>
			<td >1933078</td>
			<td >Generating a density gradient.</td>
		</tr>
		<tr >
			<td >2 M Glucose</td>
			<td >Sigma</td>
			<td >16301</td>
			<td >Generating a density gradient.</td>
		</tr>
		<tr >
			<td >H2O UltraPure Water (UPW)</td>
			<td >Bio-Lab</td>
			<td >2321777500</td>
			<td >DNase &#38; RNase free</td>
		</tr>
		<tr >
			<td >S30-T7 lysate</td>
			<td >_</td>
			<td >_</td>
			<td >Prepared at step 1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Source of transcription &#38; translation components. 
			Store at -80 &#176;c, thaw on crashed ice just before usage.&#160;</td>
		</tr>
		<tr >
			<td >Stock of DNA plasmid of choice</td>
			<td >_</td>
			<td >_</td>
			<td >Contains the sequence for the requested protein. 
			Under T7 promotor</td>
		</tr>
		<tr >
			<td colspan="4" >D. Equipment required for step 4 (synthetic cells preparation)</td>
		</tr>
		<tr >
			<td rowspan="2" >Floor incubator shaker or Thermomixer</td>
			<td >MRC</td>
			<td >TOU-120-2</td>
			<td >Laboratory shaker incubator 450x450mm, 400rpm, 70 &#176;C</td>
		</tr>
		<tr >
			<td >PHMT Grant Bio&#160;</td>
			<td >PSC18</td>
			<td >Thermomixer</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75004270</td>
			<td >(75003629) - TX-400 4 x 400mL Swinging Bucket Rotor. 
			Suited for 15 mL sized tubes. 
			Preferably swinging buckets. 
			Should enable at least 1000 x g. 
			Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >Table centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75002420</td>
			<td >(75003424) - 24 x 1.5/2.0mL rotor with ClickSeal. 
			Suited for Eppendorf vials. 
			Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td >Scientific industries</td>
			<td >SI-0256</td>
			<td ></td>
		</tr>
		<tr >
			<td >Crushed ice bucket</td>
			<td >Bel-Art</td>
			<td >M18848-4001</td>
			<td ></td>
		</tr>
		<tr >
			<td >2 mL screw neck glass vials</td>
			<td >CSI Analytical Innovations</td>
			<td >VT009M-1232</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterile 15 mL plastic tubes</td>
			<td >Thermo Scientific</td>
			<td >339651</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized 1.5 mL plastic tubes</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized pipette tips</td>
			<td >Corning</td>
			<td ></td>
			<td >Sterilized by autoclave.</td>
		</tr>
	</tbody>
</table>

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.

We present a protocol for the preparation of synthetic cells by encapsulating a S30-T7 CFPS system based on BL21 E. coli inside lipid vesicles. A schematic description of the preparation process that includes an image of each stage is presented in Figure 2. The success of the synthetic cell preparation process is dependent on the appropriate performance of each stage and effected by different parameters. The protocol should be adjusted to accommodate the production of a specific pro...

Watch this Scientific Journal Video about Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer at JoVE.com

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

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 ...

Protein producing synthetic cells offer a versatile platform for studying the origins of life as well as synthetic cell-based therapies for advanced drug delivery. This method is simple and affordable for RNA and protein expression and can be applied for onsite therapeutic protein production directly inside the body. The systems can be used for treating a wide range of diseases from metabolic diseases and protein replacements to cancer and possibly diabetes.This multi-step protocol has a defined stopping point. When performing it for the first time, it is recommended to divide the work over several days. The protocol for synthetic cell production starts with the preparation of the cell-free S30-T7 lysate.Next, the membrane lipids and cellular nutrient solution are prepared to simulate the inner environment and to support protein production. The final step is the preparation of the synthetic cells themselves. Prepare duplicate starter cultures in 100 milliliter Erlenmeyer flasks.To each flask, add five milliliters of LB media supplemented with 50 micrograms per milliliter of ampicillin and inoculate with a single E.coli colony. Grow the culture overnight in a floor incubator shaker at 250 rpm and 37 degrees Celsius. Next, prepare two liter baffled Erlenmeyer flasks containing 500 milliliters of TB media supplemented with 50 micrograms per milliliter of ampicillin.Inoculate each two liter flask with a five milliliter starter culture. Place the flasks in a shaker at 250 rpm and 37 degrees Celsius. Monitor the cultures periodically using a spectrophotometer and grow the cultures until OD600 between 0.8 and one.To induce T7 RNA polymerase expression, add to each flask three milliliters of 100 millimolar IPTG to reach 0.6 millimolar. Continue growing the cultures until OD600 is approximately four. After the cultures reach the desired optical density, transfer the suspension from each Erlenmeyer flask into two 250 milliliter sterilized centrifuge tubes.Centrifuge the tubes at 7, 000 times g for 10 minutes at four degrees Celsius. Discard the supernatant. Resuspend each pellet in 250 milliliters of cold S30 lysate buffer using a stirrer if desired.Centrifuge at 7, 000 times g for 10 minutes at four degrees Celsius. Discard the supernatant and resuspend all the pellets together in 15 milliliters of cold S30 lysate buffer. Filter the suspension using gauze pads.Before proceeding to the next step, precool the tips in 1.5 milliliter vials that will be needed for storing the lysate. Homogenize the suspension twice. Use a working pressure of 15, 000 PSI with an air pressure of four bar for cell breakage.Collect the homogenate. Add 100 microliters of 0.1 molar DTT per 10 milliliters of the homogenized suspension. Centrifuge the suspension at 24, 700 times g at 30 minutes at four degrees Celsius.DTT and chloroform are classified as irritant and harmful and should therefore be treated with care. Chloroform used later in the protocol must be used in an area with fume extraction. To preserve the lysate activity, perform the step quickly.Place 200 microliter aliquots of supernatant in pre-cooled vials and immediately snap freeze them with liquid nitrogen. Store the vials at negative 80 degrees Celsius for future use. Separately dissolve POPC and cholesterol in chloroform each to a final concentration of 100 milligrams per milliliter.Vortex each vial separately. Combine the components in a two milliliter glass vial. Add 50 microliters of the cholesterol-chloroform solution, 50 microliters of the POPC-chloroform solution, and 500 microliters of mineral oil.Vortex the vial, then to evaporate the chloroform, heat it for about one hour at 80 degrees Celsius in a chemical hood. Before starting the synthetic cell production stage, prepare the outer, pre-inner, and feeding solutions as described in the manuscript. Then place 1.2 milliliters of the outer solution in a 15 milliliter tube and slowly add a layer of 500 microliters of lipids-in-oil solution from the first glass vial.Incubate the tube at room temperature for 20 minutes. To finalize the inner solution preparation, mix on ice to a final volume of 100 microliters by adding S30-T7 lysate and DNA plasmid. Add 100 microliters of the inner solution with lysate and plasmid to the second two milliliter glass vial with 500 microliters of lipids-in-oil solution.Pipette up and down vigorously for one minute and then vortex for another minute on level five. After incubating the resulting emulsion for 10 minutes on crushed ice, slowly add the emulsion on top of the oil phase in the 15 milliliter tube. Centrifuge the tube for 10 minutes at 100 times g and four degrees Celsius.Then centrifuge for 10 minutes at 400 times g and four degrees Celsius. By the end of the centrifugation, a pellet should be observable at the bottom of the tube. To extract the pellet, first remove the excess oil layer.Next, load a trimmed pipette tip with approximately 400 microliters of outer solution and use the pipette tip to collect the pellet, releasing the outer solution as the pipette tip passes through the oil phase. Wipe the pipette tip and transfer the pellet to a clean 1.5 milliliter tube. Centrifuge the pellet for 10 minutes at 1, 000 times g and four degrees Celsius.Remove the supernatant and resuspend the pellet in 100 microliters of feeding solution. For protein expression, incubate the suspension for two hours at 37 degrees Celsius without shaking. Plasmids expressing the model protein sfGFP and Renilla luciferase were introduced into cell-free protein synthesis, CFPS bulk reactions, and synthetic cells.Protein production was evaluated using several methods. Western blot analysis detected sfGFP His6 production in both CFPS bulk reactions and inside synthetic cells. Purified sfGFP His6 was used as a positive control.A fluorescent microscope with a Brightfield and a GFP filter show synthetic cells that are producing sfGFP. Flow cytometry was used to analyze sfGFP producing synthetic cells. Calculation of the active synthetic cell population was based on the sfGFP fluorescence intensity threshold defined by the negative DNA sample represented by the red histogram.Mean fluorescence intensity of sfGFP producing synthetic cells was calculated from the active synthetic cell population and normalized to the negative DNA sample. The diameter distributions of the active synthetic cells were calculated based on the sfGFP signal. Renilla luciferase activity was quantified with luminescence measurements.This method is highly versatile and can be optimized for different target proteins by altering lysate origin, lipid composition and inner solution concentrations. FACS analysis of synthetic cells producing a fluorescent marker protein could be performed to provide quantitative value of the fraction of active synthetic cells. Western blot analysis would measure protein production.

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

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

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 ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

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 ...

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 ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

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 ...