Name: escherichia coli-based cell-free protein synthesis: protocols for a robust, flexible, and accessible platform technology
Uploaded: 2019-02-25T12:30:00
Description: Watch this Scientific Journal Video about Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology at JoVE.com

Authors would like to acknowledge Dr. Jennifer VanderKelen, Andrea Laubscher, and Tony Turretto for technical support, Wesley Kao, Layne Williams, and Christopher Hight 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 Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF-1708919). MZL acknowledges the California State University Graduate Grant. MCJ acknowledges the Army Research Office W911NF-16-1-0372, National Science Foundation grants MCB-1413563 and MCB-1716766, the Air Force Research Laboratory Center of Excellence Grant FA8650-15-2-5518, the Defense Threat Reduction Agency Grant HDTRA1-15-10052/P00001, the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, the Department of Energy BER Grant DE-SC0018249, the Human Frontiers Science Program (RGP0015/2017), the DOE Joint Genome Institute ETOP Grant, and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust for support.

1. Media Preparation and Cell Growth
<ol>
	<li>Day 1
	<ol>
		<li>Streak E. coli BL21*(DE3) cells from a glycerol stock onto an LB agar plate and incubate for at least 18 h at 37 &#176;C.</li>
		<li>Prepare 50 mL of LB media and autoclave the solution on a liquid cycle for 30 min at 121 &#176;C. Store at room temperature.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Prepare 750 mL of 2x YTP media and 250 mL of 0.4 M D-Glucose solution as described in the supplemental information.<...

Cell-free protein synthesis has emerged as a powerful and enabling technology for a variety of applications ranging from biomanufacturing to rapid prototyping of biochemical systems. The breadth of applications is supported by the capacity to monitor, manipulate, and augment cellular machinery in real-time. In spite of the expanding impact of this platform technology, broad adaptation has remained slow due to technical nuances in the implementation of the methods. Through this effort, we aim to provide simplicity and cla...

Cell-free Protein Synthesis (CFPS) has emerged as a technology that has unlocked a number of new opportunities for protein production, functional genomics, metabolic engineering, and more within the last 50 years1,2. Compared to standard in vivo protein expression platforms, CFPS provides three key advantages: 1) the cell-free nature of the platform enables the production of proteins that would be potentially toxic or foreign to the cell3,4,5,6; 2) inactivation of genomic DNA and the introduction of a template DNA encoding the gene(s) of interest channel all of the systemic energy within the reaction to the production of the protein(s) of interest; and 3) the open nature of the platform enables the user to modify and monitor the reaction conditions and composition in real time7,8. This direct access to the reaction supports the augmentation of biological systems with expanded chemistries and redox conditions for the production of novel proteins and the tuning of metabolic processes2,9,10. Direct access also allows the user to combine the CFPS reaction with activity assays in a single-pot system for more rapid design-build-test cycles11. The capacity to perform the CFPS reaction in small volume droplets or on paper-based devices further supports high-throughput discovery efforts and rapid prototyping12,13,14,15,16. As a result of these advantages and the plug and play nature of the system, CFPS has uniquely enabled a variety of biotechnology applications such as the production of proteins that are difficult to solubly express in vivo17,18,19,20, detection of disease21,22,23, on demand biomanufacturing18,24,25,26,27, and education28,29, all of which show the flexibility and utility of the cell-free platform.
CFPS systems can be generated from a variety of crude lysates from both prokaryotic and eukaryotic cell lines. This allows for diverse options in the system of choice, each of which have advantages and disadvantages depending on the application of interest. CFPS systems also vary greatly in preparation time, cost, and productivity. The most commonly utilized cell extracts are produced from wheat germ, rabbit reticulocyte, insect cells, and Escherichia coli cells, with the latter being the most cost-effective to date while producing the highest volumetric yields of protein30. While other CFPS systems can be advantageous for their innate post-translational modification machinery, emerging applications using the E. coli-based machinery are able to bridge the gap by generating site-specifically phosphorylated and glycosylated proteins on demand31,32,33,34,35.
CFPS reactions can be run in either batch, continuous-exchange cell-free (CECF) or continuous-flow cell-free (CFCF) formats. The batch format is a closed system whose reaction lifetime is limited due to diminishing quantities of reactants and the accumulation of inhibitory byproducts of the reaction. CECF and CFCF methods increase the lifetime of the reaction, and thereby result in increased volumetric protein yields compared to the batch reaction. This is accomplished by allowing the byproducts of protein synthesis to be removed from the reaction vessel while new reactants are supplied throughout the course of the reaction2. In the case of CFCF, the protein of interest can also be removed from the reaction chamber, while in CECF, the protein of interest remains in the reaction chamber comprised of a semi-permeable membrane36,37. These methods are especially valuable in overcoming poor volumetric yields of difficult-to-express proteins of interest38,39,40,41,42,43. The challenges in implementing the CECF and CFCF approaches are that 1) while they result in more efficient use of the bio machinery responsible for transcription and translation, they require notably larger quantities of reagents that increases overall cost and 2) they require more complex reaction setups and specialized equipment compared to the batch format44. In order to ensure accessibility for new users, the protocols described herein focus on the batch format at reaction volumes of 15 &#181;L with specific recommendations for increasing the reaction volume to the milliliter scale.
The methods presented herein enable non-experts with basic laboratory skills (such as undergraduate students) to implement cell growth, extract preparation, and batch format reaction setup for an E. coli-based CFPS system. This approach is cost-effective compared to commercially available kits without sacrificing the ease of kit-based reaction setup. Furthermore, this approach enables applications in the laboratory and in the field. When deciding to implement CFPS, new users should thoroughly evaluate the efficacy of conventional protein expression systems for startup investment, as CFPS may not be superior in every case. The CFPS methods described here enable the user to directly implement a variety of applications, including functional genomics, high-throughput testing, the production of proteins that are intractable for in vivo expression, as well as field applications including biosensors and educational kits for synthetic biology. Additional applications such as metabolic engineering, tuning of protein expression conditions, disease detection, and scale-up using CECF or CFCF methods are still possible but may require experience with the CFPS platform for further modification of reaction conditions. Our methods combine growth in enriched media and baffled flasks, with relatively rapid and reproducible methods of cell lysis through sonication, followed by a simplified CFPS reaction setup that utilizes optimized premixes45. While the cellular growth methods have become somewhat standardized within this field, methods for cell lysis vary widely. In addition to sonication, common lysis methods include utilization of a French press, a homogenizer, bead beaters, or lysozyme and other biochemical and physical disruption methods46,47,48,49. Using our methods, approximately 2 mL of crude cell extract are obtained per 1 L of cells. This quantity of cell extract can support four hundred 15 &#181;L CFPS reactions, each producing ~900 &#181;g/mL of reporter sfGFP protein from the template plasmid pJL1-sfGFP. This method costs $0.021/&#181;g of sfGFP produced ($.019/&#181;L of reaction), excluding the cost of labor and equipment (Supplemental Figure 1). Starting from the scratch, this method can be implemented in 4 days by a single person and repeat CFPS reactions can be completed within hours (Figure 1). Additionally, the protocol can be scaled up in volume for larger batches of reagent preparation to suit the user&#39;s needs.Importantly, the protocol presented here can be implemented by laboratory trained non-experts such as undergraduate students, as it only requires basic laboratory skills. The procedures described below, and the accompanying video have been specifically developed to improve accessibility of the E. coli CFPS platform for broad usage.

<table border="0" cellpadding="0" cellspacing="0" style="width:817px;" width="817">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Luria Broth</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:123px;">12795027</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Tryptone</td>
			<td style="width:145px;">Fisher Bioreagents</td>
			<td style="width:123px;">73049-73-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Yeast Extract</td>
			<td style="width:145px;">Fisher Bioreagents</td>
			<td style="width:123px;">1/2/8013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NaCl</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">S3014-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Potassium Phosphate Dibasic</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">60353-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Potassium Phosphate Monobasic</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P9791-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">D-Glucose</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">KOH</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P5958-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IPTG</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Mg(OAc)2</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">M5661-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">K(OAc)</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P1190-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tris(OAc)</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">T6066-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DTT</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:123px;">15508013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">tRNA</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">10109541001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Folinic Acid</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">F7878-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NTPs</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:123px;">R0481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Oxalic Acid</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P0963-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NAD</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">N8535-15VL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">CoA</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">C3144-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PEP</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">860077-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">K(Glu)</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">G1501-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">NH4(Glu)</td>
			<td style="width:145px;">MP Biomedicals</td>
			<td style="width:123px;">02180595.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Mg(Glu)2</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">49605-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Spermidine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">S0266-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Putrescine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">D13208-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">HEPES</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:123px;">11344041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Molecular Grade Water</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">7732-18-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Aspartic Acid</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">A7219-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Valine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">V0500-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Tryptophan</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">T0254-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Phenylalanine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P2126-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Isoleucine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">I2752-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Leucine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">L8000-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Cysteine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">C7352-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Methionine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">M9625-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Alanine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">A7627-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Arginine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">A8094-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Asparagine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">A0884-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Glycine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">G7126-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Glutamine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">G3126-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Histadine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">H8000-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Lysine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">L5501-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Proline</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">P0380-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Serine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">S4500-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Threonine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8625-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L-Tyrosine</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">T3754-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Fisherbrand Premium Microcentrifuge Tubes: 2.0 mL</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">05-408-138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fisherbrand Premium Microcentrifuge Tubes: 0.6 mL</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">05-408-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">PureLink HiPure Plasmid Prep Kit</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:123px;">K210007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ultrasonic Processor</td>
			<td style="width:145px;">QSonica</td>
			<td style="width:123px;">Q125-230V/50Hz</td>
			<td>3.175 mm diameter probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Avanti J-E Centrifuge</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:123px;">369001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">JLA-8.1000 Rotor</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:123px;">366754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1L Centrifuge Tube</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:123px;">A99028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tunair 2.5L Baffeled Shake Flask</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:123px;">Z710822</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microfuge 20</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:123px;">B30134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">New Brunswick Innova 42/42R Incubator</td>
			<td style="width:145px;">Eppendorf</td>
			<td style="width:123px;">M1335-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cytation 5</td>
			<td style="width:145px;">BioTek</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Strep-Tactin XT Starter Kit</td>
			<td style="width:145px;">IBA</td>
			<td style="width:123px;">2-4998-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">pJL1-sfGFP</td>
			<td style="width:145px;">Addgene</td>
			<td style="width:123px;">69496</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">BL21(DE3)</td>
			<td style="width:145px;">New England BioLabs</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
	</tbody>
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escherichia coli-based cell-free protein synthesis: protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

We have presented a sonication-based E. coli extract preparation protocol that can be completed over a four-day span, with Figure 1 demonstrating the procedural breakdown over each day. There is malleability to the steps that can be completed in each day with various pausing points, but we have found this workflow to be the most effective to execute. Additionally, both the cell pellets (step 1.3.18) and fully prepared extract (step 2.10) are stable a...

Watch this Scientific Journal Video about Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology at JoVE.com

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

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