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 to below 40 &#176;C after autoclaving.</li>
	<li>Prior to inoculation of the CFAI media, add the sugar solution directly to the CFAI media.</li>
	<li>To inoculate the media, swipe a loopful of colonies from a previously streaked E. coli BL21 Star (DE3) plate and insert directly into the media. Swirl the loop into the media but avoid touching the sides of the container. Ensure that the streak plate is fresh, with viable cells.</li>
	<li>Place the flask with the inoculated media in a 30 &#176;C incubator while shaking at 200 rpm. Allow the culture to grow overnight. If cells are inoculated in the evening, the culture will reach an approximate optical density, measured at 600 nm (OD600), of 10 the subsequent morning. Inoculation and harvest times can be adjusted as needed.</li>
</ol>
2. Cell harvest
<ol>
	<li>Prepare S30 buffer in advance and keep it cold. Prepare the S30 buffer according to Table 2, to a final pH of 8.2. 
	NOTE: The S30 buffer can be prepared days prior to the cell harvest. If this is done, prepare without dithiothreitol and store at 4 &#176;C. Add dithiothreitol just before use.</li>
	<li>From this point forward, keep all solutions and materials on ice. Transfer 1 L of media into a 1 L centrifuge bottle and centrifuge at 5,000 x g between 4-10 &#176;C for 10 min. Decant and dispose of the supernatant. Using a sterile spatula, transfer the pellet to a pre-chilled and previously weighed 50 mL conical tube.</li>
	<li>Wash once with 30-40 mL of cold S30 buffer by resuspending the pellet via vortexing in 30 s bursts with rest periods on ice. 
	NOTE: Due to a higher volume of pellet, splitting the cell pellet into two 50 mL conical tubes can be helpful for the wash step. Storing cells in smaller aliquots also provides flexibility for the downstream processing.</li>
	<li>Centrifuge the cell resuspension at 5000 x g between 4-10 &#176;C for 10 min.</li>
	<li>Dispose of the supernatant and wipe any excess from the inside walls of the 50 mL conical tube using a clean tissue, avoid touching the pellet itself. Weigh and flash freeze the pellet in liquid nitrogen. Store at -80 &#176;C until further use. 
	&#8203;NOTE: Pellets may not require flash-freezing if the user plans to continue with the extract preparation protocol.</li>
</ol>
3. Extract preparation
<ol>
	<li>Combine the frozen pellet with 1 mL of S30 buffer for every 1 g of the cell pellet and allow to thaw on ice for approximately 30-60 min. Resuspend thawed pellet via vortexing in bursts of 30 s with rest periods on ice. Vortex until there are no visible clumps of cells remaining. 
	NOTE: Smaller clumps can be resuspended by mixing using a pipette.</li>
	<li>Transfer aliquots of 1.4 mL of cell resuspension into 1.5 mL microfuge tubes for cell lysis. Sonicate each tube with a frequency of 20 kHz and 50% amplitude for three bursts of 45 s with 59 s of rest per cycle surrounded by an ice bath. Invert the tube between cycles and immediately add 4.5 &#181;L of 1 M dithiothreitol after the last sonication cycle. 
	NOTE: Due to the heat released from sonication, it is extremely important to make sure all aliquots are kept on ice when not being sonicated. The ice bath should be constantly replenished or large enough to stay cool throughout the entire sonication process.</li>
	<li>Centrifuge each tube at 18,000 x g and 4 &#176;C for 10 min. Retrieve the supernatant and aliquot into 1.5 mL microfuge tubes in 600 &#181;L aliquots. Flash freeze aliquots and store at -80 &#176;C until further use. Care should be taken to pipette only the supernatant.</li>
</ol>
4. Cell-free protein synthesis
<ol>
	<li>Thaw one aliquot of extract from the previous step to perform 15 &#181;L of cell-free protein synthesis reactions in 1.5 mL microfuge tubes in quadruplicate.</li>
	<li>Prepare each reaction by combining 240 ng of DNA (16 &#181;g/mL final concentration), 2.20 &#181;L of Solution A, 2.10 &#181;L of Solution B, 5.0 &#181;L of extract, and a varying volume of molecular-grade water to fill the reaction to 15 &#181;L. This reaction can be scaled to higher volumes. See Table 3 for ratios.
	<ol>
		<li>Prepare Solution A and B according to Table 4. Each solution can be prepared in batches of 100 &#181;L to 1 mL, aliquoted, and stored at -80 &#176;C until further use. 
		NOTE: The DNA amount can vary depending on the protein of interest. In this case, the plasmid used, pJL1-sfGFP, has been optimized to perform at 16&#160;&#181;g/mL, or 597 &#181;M.</li>
	</ol>
	</li>
	<li>Let the reactions run for at least 4 h at 37 &#176;C.</li>
</ol>
5. Quantification of reporter protein, super folder green fluorescent protein (sfGFP)
<ol>
	<li>Using a half area 96-well black polystyrene plate, combine 2 &#181;L of each cell-free protein synthesis reaction product with 48 &#181;L of 0.05 M HEPES buffer at pH 7.2. Three to four replicates of each reaction tube are recommended.</li>
	<li>Quantify the fluorescence intensity of the sfGFP with an excitation wavelength of 485 nm and an emission wavelength of 528 nm.</li>
	<li>For conversion of relative fluorescence units to volumetric yield (&#181;g/mL) of sfGFP, establish a standard curve using purified pJL1-sfGFP.</li>
</ol>

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The authors declare that they have no competing financial conflicts of interest.

Researcher oversight is traditionally needed for two key actions during cell growth: the induction of T7 RNAP and harvesting cells at a specific OD600. CFAI obviates both of those requirements to decrease the researcher&#39;s time and technical training required in order to prepare high quality cell extracts. Auto-induction of T7 RNAP is achieved by replacing glucose with lactose as the primary sugar in the media, obviating the previous need to actively monitor the growth and then induce with IPTG&#160;at a pr...

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

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

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

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

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

from cells to cell-free protein synthesis within 24 hours using cell-free autoinduction workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

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

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

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

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

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Research

JoVE Journal

Bioengineering

4.2K Views.  California Polytechnic State University San Luis Obispo. 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. 

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

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

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

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

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

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

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

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

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

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

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

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

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

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.

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 ...

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

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

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

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

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

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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 in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.