We thank Jongmin Kim, Dan Siegal-Gaskins, Anu Thubagere, and Enoch Yeung for assistance streamlining the protocol, and Clare Chen and Barclay Lee for assistance in the early stages of the project. This material is based upon work supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 (DARPA/CMO.Z.Z.S. is also supported by a UCLA/Caltech Medical Scientist Training Program fellowship and by a DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.

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The authors declare that they have no competing financial interests.

The endogenous Escherichia coli based TX-TL cell-free expression system described here is an easy-to-run three tube reaction that can take less than eight hours from set up to data collection. The process of creating all reagents requires five days' time total (with significant labor requirements on only one day), but produces crude extract for 3,000 reactions and buffer-making reagents for 10,000 reactions (Figure 1). Furthermore, crude extract and buffer-making reagents are stable for at least 1 year at -80 &deg;C, allowing for multiple uses of one preparation.4 At $0.11 per 10 &mu;l reaction ($0.26 including labor), costs are 98% lower than comparable commercial systems (Figure 2). 
There are some unresolved limitations, however, to the system. The end efficiency of each crude cell extract preparation can vary based on user proficiency and on environmental conditions, although typical yield variation is between 5-10% (Figure 4b). As a result, batch-to-batch variability in both end-point expression and in expression dynamics are to be expected. These variations will likely remain until extract is fully characterized or until extract creation is fully automated. If the cell-free expression system is used to conduct sensitive quantitative experiments, it is advisable to run all experiments with the same batch of crude cell extract. The yield from a single crude cell extract batch, about 3000 reactions, should be sufficient for typical experimental courses. Although we suspect variation can be removed by scaling up and automating the procedure, such attempts would involve a substantial resource investment. 
Additionally, although end-point expression levels are reasonably easy to determine, more work needs to be done in understanding dynamics intrinsic to the cell-free system. It is known that both resource competition and resource limitation can affect expression dynamics. For example, limited endogenous sigma 70 can result in a saturating regime with increased DNA template producing an expression profile analogous to that of nucleotide or amino acid depletion.9,27 However, dynamics do not have to be fully understood to utilize the system. For pure increases of yield, optimization can be done by machine-learning approaches.28 Questions of resource competition and limitation can be addressed by mathematical models verified using experimental data.
The protocol presented here is optimized for a BL21-Rosetta2 strain, but is generalizable to other E. coli strains. Modifications in BL21-Rosetta2, such as the removal of the gene encoding lon protease and the addition of genes encoding rare tRNAs, allow for maximal protein production. We have attempted the protocol with two other extract strains - BL21 only and a BL21 trxA knockout -and found 50% less protein yield. We hypothesize that yields similarly decrease when using other strains. Other changes in parameters, such as switching 2xYT growth medium for LB and other rich broths, have resulted in decreased protein yield. 
Cell-free expression systems utilizing both endogenous and exogenous transcription-translation machinery and regulation mechanisms have wide applications in both protein and metabolite expression and in synthetic biology.3,29 Instead of being limited to T7-regulated circuits, one can envision producing complex biomolecules in a user-controllable setting using a mix of native E. coli promoters and exogenously supplied transcription and regulation mechanisms. Without limitations of cell division and metabolism, variability in synthetic circuits such as the repressilator or in metabolic engineered pathways such as those producing artemisinin can be reduced or better understood.30,31 We have used these advantages to implement genetic switches, as well as to understand sigma factor sequestration.9,32 Such technology can also form the backbone of "minimal" or "artificial" cells - small, well-characterized and self-sufficient embodied units of extract.33,34 
Ultimately, we anticipate immediate uses of this endogenous cell-free expression system as a prototyping environment for synthetic biology. Nicknamed the "TX-TL biomolecular breadboard," the cell-free expression system provides a controllable environment where synthetic circuits ultimately destined for in vivo expression can undergo rounds of prototyping - cycles of testing on basic plasmid, linear, or chemically synthetized DNA, followed by analysis and rapid modification. Prototyping rounds can be aided by predictive mathematical models currently being developed. By removing cloning and in vivo manipulation for non-final circuits, we anticipate engineering cycle times to be reduced to 1-3 days instead of the current weeks' standard.

A correction to Figure 5's legend has been made for the article <a href="http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free">Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology</a>. Method 1 and method 2 have been switched.Z
The figure legend was update from:
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1 nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 1) or post-processed with a QiaQuick PCR purification kit (Purification method 2).
to
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1
nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 2) or post-processed
with a QiaQuick PCR purification kit (Purification method 1).

A correction to Figure 5's legend has been made for the article Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Method 1 and method 2 have been switched.

Erratum: Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Cell-free expression technology began in the 1950s as purely translational, advancing years later to encompass coupled transcription-translation mechanisms using T7 bacteriophage DNA.11,12 Since then, numerous efforts have been made to optimize the creation of crude cell extract (or E. coli S30 extract).13,14 These optimizations include prolonging cell-free protein synthesis through ATP regeneration or strain modifications, and reducing protocol time and cost.15-17 Alternative cell-free expression systems exist that use reconstituted components in lieu of crude cell extract for expression.5 Both crude cell extract and reconstitution methods have been developed for commercial use.
With the advent of synthetic biology, there is an increased need for a well-characterized platform to test and express engineered biological modules and circuits.18,19 This platform must be versatile, well-characterized, simple to manipulate, and focused on user-supplied components. Despite being developed half a century earlier, cell-free systems based on E. coli intrinsically share these requirements, as they are a simplified in vitro representation of cellular processes without the complexity of growth and metabolism. Additionally, all of the foundational knowledge from in vivo work on E. coli applies readily to E. coli cell-free systems.
Although cell-free expression systems can have applications in synthetic biology, to date the goal of most cell-free expression systems has been the maximization of protein and metabolite yield. This is accomplished by using T7 bacteriophage transcription of sequences driven by T7 promoters.20 Although expression is efficient and robust, these systems serve a highly specialized purpose. Cell regulation methods are limited, target DNA templates must be reengineered to include T7 promoters, and certain sequences such as ribosomal complexes cannot be transcribed and assembled.21,22 Existing cell-free expression systems are unable to maintain high yields while preserving endogenous regulatory mechanisms, a versatility necessary for synthetic biology.
 We have developed an endogenous E. coli cell-free expression system that preserves the efficiency of protein expression demonstrated by previous systems but adds additional versatility by allowing expression and regulation based on both endogenous and exogenous (T7 or other) mechanisms. The protocol described here is originally based on Kigawa et al. (2004) and Liu et al. (2005), but has significant modifications. It utilizes Mg- and K- glutamate over Mg- and K- acetate for increased efficiency, removes 2-mercaptoethanol, and lyses cells using a bead-beater.17,23,24 Bead-beating is chosen over homogenization, pressure-based methods, or sonication due to its lower cost and comparable yields to competing systems.23 3-phosphoglyceric acid (3-PGA) is used as the energy source as it was found to give superior protein yields when compared to creatine phosphate and phosphoenolpyruvate.4,25 Our system can produce up to 0.75 mg/ml of reporter protein using either a sigma70-based promoter with lambda-phage operators or a T7-driven promoter, similar to yields from other commercial systems.4,6 Five days are required to produce all necessary reagents (Figure 1). Furthermore, it provides a 98% cost reduction compared to comparable commercial cell-free systems - material costs are $0.11 per 10 &mu;l reaction, which rises to $0.26 with labor included (Figure 2).

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>2xYT</td>
 <td>MP biomedicals</td>
 <td>3012-032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3-PGA</td>
 <td>Sigma-Aldrich</td>
 <td>P8877</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A8937</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bacto-agar</td>
 <td>BD Diagnostics</td>
 <td>214010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bead-beating tubes (polypropylene microvials)</td>
 <td>BioSpec</td>
 <td>522S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Beads, 0.1mm dia.</td>
 <td>BioSpec</td>
 <td>11079101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BL21 Rosetta 2 E. coli strain</td>
 <td>Novagen</td>
 <td>71402</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bradford BSA Protein Assay Kit</td>
 <td>Bio-rad</td>
 <td>500-0201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cAMP</td>
 <td>Sigma-Aldrich</td>
 <td>A9501</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C1919</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CoA</td>
 <td>Sigma-Aldrich</td>
 <td>C4282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CTP</td>
 <td>USB</td>
 <td>14121</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cuvettes, 1.5ml</td>
 <td>Fisher</td>
 <td>14-955-127</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma-Aldrich</td>
 <td>D0632</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Folinic acid</td>
 <td>Sigma-Aldrich</td>
 <td>F7878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GTP</td>
 <td>USB</td>
 <td>16800</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H6147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>K-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>G1149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mg-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>49605</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro Bio-Spin Chromatography Columns</td>
 <td>Bio-Rad</td>
 <td>732-6204</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NAD</td>
 <td>Sigma-Aldrich</td>
 <td>N6522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc 384-well optical bottom plates</td>
 <td>Thermo-Scientific</td>
 <td>142761</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc sealing tape</td>
 <td>Thermo-Scientific</td>
 <td>232701</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PEG-8000</td>
 <td>Promega</td>
 <td>V3011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate dibasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8584</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate monobasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8709</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RTS Amino Acid Sampler</td>
 <td>5 Prime</td>
 <td>2401530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit)</td>
 <td>Thermo-Scientific</td>
 <td>66382</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spermidine</td>
 <td>Sigma-Aldrich</td>
 <td>85558</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base</td>
 <td>Fischer</td>
 <td>BP1521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA (from E. coli)</td>
 <td>Roche Applied Science</td>
 <td>MRE600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UTP</td>
 <td>USB</td>
 <td>23160</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1L Centrifuge Bottle</td>
 <td>Beckman-Coulter</td>
 <td>A98813</td>
 <td>This is specific for Avanti J-series; obtain equivalent size for centrifuge in use.</td>
 </tr>
 <tr>
 <td>4L Erlenmeyer Flask</td>
 <td>Kimble Chase</td>
 <td>26500-4000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Avanti J-26XP Centrifuge</td>
 <td>Beckman-Coulter</td>
 <td>393127</td>
 <td>Or 1L-capable centrifuge equivalent.</td>
 </tr>
 <tr>
 <td>Forma 480 Orbital Shaker</td>
 <td>Thermo Scientific</td>
 <td>480</td>
 <td>Or chest-size 6x4L shaker equivalent.</td>
 </tr>
 <tr>
 <td>JLA-8.1000 Rotor</td>
 <td>Beckman-Coulter</td>
 <td>363688</td>
 <td>Or 1L-capable, 5000 x g rotor equivalent for centrifuge.</td>
 </tr>
 <tr>
 <td>Mini-Beadbeater-1</td>
 <td>BioSpec</td>
 <td>3110BX</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
Supplemental Material 1. Recipes for Items. 
Chloramphenicol, 34 mg/ml: Prepare 0.51 g chloramphenicol and add ethanol to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
2xYT+P+Cm agar plate: Prepare 1.24 g 2xYT, 1.6 ml potassium phosphate dibasic solution @ 1 M, 0.88 ml potassium phosphate monobasic solution @ 1 M, 0.6 g agar, and water to 40 ml. Autoclave. Let cool to 50 &deg;C and add 40 &mu;l Cm. Aliquot 25 ml into a 100x15 mm petri dish, and let cool for an hour. 
2xYT+P media: Prepare 124 g 2xYT, 160 ml potassium phosphate dibasic solution @1 M, 88 ml potassium phosphate monobasic solution @ 1 M, and water to 4 L. Aliquot out into 2x1.88 L and 0.24 L. Autoclave. 
Tris base, 2 M: Prepare 60.57 g Tris base and water to 250 ml. Sterilize, store at RT for later use. 
DTT, 1 M: Prepare 2.31 g DTT and water to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
S30A buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, 50 ml Tris at 2M, acetic acid (to pH 7.7), and water to 2 L. Autoclave, store at 4 &deg;C, add 4 ml 1 M DTT before use. 
S30B buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, Tris at 2 M (to pH 8.2), and water to 2 L. Autoclave, store at 4 &deg;C, add 2 ml 1 M DTT before use. 
HEPES: Prepare 1.91 g HEPES (MW 238.21), KOH (to pH 8), and water to 4 ml. 
tRNA: Prepare 30 mg of tRNA and water to 600 &mu;l. 
CoA: Prepare 30 mg of CoA (MW 767.53) and water to 600 &mu;l. 
NAD: Add 34.83 mg of NAD (MW 663.43), Tris at 2 M (to pH 7.5-8), and water to 300 &mu;l. (Add 27 &mu;l of Tris at 2 M to bring the solution to pH 7.5-8). 
cAMP: Add 42.80 mg of cAMP (MW 329.22), Tris at 2 M (to pH 8), and water to 200 &mu;l. (Add 73 &mu;l of Tris at 2 M to bring the solution to pH 8). 
Folinic Acid (33.9 mM): To 20 mg of solid folinic acid calcium salt (MW 511.5), add 1.15 ml water. 
Spermidine: Prepare 23.55 &mu;l of spermidine (MW 145.25) and water to 150 &mu;l. Prepare at room temperature after melting briefly at 37 &deg;C. 
3-PGA: Add 1.03 g of 3-PGA (MW 230.02), Tris at 2 M (to pH 7.5), and water to 3.2 ml. (Add 1.73 ml of Tris at 2 M to bring the solution to pH 7.5). 
Nucleotide Mix: Add 145 mg of ATP dipotassium salt dihydrate (MW 619.4), 133 mg of GTP disodium salt (MW 567.14), 79.4 mg of CTP disodium salt dihydrate (MW 563.16), 82.6 mg of UTP trisodium salt dihydrate (MW 586.12), KOH at 15% dilution (to pH 7.5), and water to 1.5 ml. (Add 353 &mu;l of KOH at 15% dilution to bring the solution to pH 7.5). 
Supplemental Material 2. Bradford Assay.
<ol>
<li>Remove Bradford agent from 4 &deg;C and set at room temperature.</li>
<li>Prepare 50 &mu;l BSA Standard at 1 mg/ml and at 0.1 mg/ml.</li>
<li>Prepare 40 &mu;l 20x dilution of extract from step 1.47.</li>
<li>Add 800 &mu;l water to 7 cuvettes. </li>
<li>Prepare standard cuvettes for 0 mg/ml, 1 mg/ml (10 &mu;l 0.1 mg/ml BSA), 2 mg/ml (20 &mu;l 0.1 mg/ml BSA), 4 mg/ml (4 &mu;l 1 mg/ml BSA), 6 mg/ml (6 &mu;l 1 mg/ml BSA).</li>
<li>Prepare experimental cuvettes for 2 &mu;l of sample and 4 &mu;l of sample.</li>
<li>Add 200 &mu;l of Bradford agent to each cuvette and mix well by pipetting. Incubate at room temperature for at least 10 min.</li>
<li>Produce standard curve at OD 595nm using cuvettes from step 6.5. Reject standard curve if r2 &lt; 0.95.</li>
<li> Determine extract concentration at OD 595nm using cuvettes froms tep 6.6.</li>
</ol>
Supplemental Material 3. Buffer calibration spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank">See TXTL_e(template)_calibration_JoVE.xlsx</a>.
 
Supplemental Material 4. Cell-free expression run spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">See TXTL _JoVE.xlsx</a>.
 </td>
 </tr>
 </tbody>
</table>

protocols for implementing an escherichia coli based tx-tl cell-free expression system for synthetic biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

We have presented a five day protocol for the preparation of an endogenous Escherichia coli based TX-TL cell-free expression system. A sample timeline for creating the reagents - crude cell extract and buffer - can be found in Figure 1. Once created, these can be stored at -80 &deg;C for up to one year. After reagents are created, experimental setup and execution can be done in less than 8 hr.
In addition, we optimized the expression conditions of the TX-TL cell-free expression system. Other user-supplied additions, such as buffers or DNA solutions, should be calibrated for toxicity beforehand. For example, different methods of processing plasmids result in different expression due to salt content. We also tested the effect of Tris-Cl elution buffer on reaction efficiency (Figure 5).
An example of crude cell extract calibration, referring step 4.1 to 4.9, is shown in Figure 4a. In general, our experiments show that the crude cell extract is most sensitive to Mg-glutamate levels, followed by K-glutamate levels. To demonstrate the cell-free expression system, we constructed and tested a negative feedback loop based on tet repression.26 (Figure 6). In the cell-free expression system, the same circuit run with and without aTc shows a 7-fold end-point expression change of deGFP reporter after eight hours of expression. Although this experiment does not require global inducers or repressors, if necessary they can be added under "Master Mix Preparation."
<img alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50762/50762fig1highres.jpg" src="/files/ftp_upload/50762/50762fig1.jpg" /> 
Figure 1. Timeline for crude cell extract, amino acid solution, and energy solution preparation. A five-day timeline for a typical execution of the protocol is given above, optimized for overnight incubations and daytime working steps.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig2highres.jpg" src="/files/ftp_upload/50762/50762fig2.jpg" /> 
Figure 2. Cost and expression analysis of competing crude cell extracts. a) Breakdown of the costs of labor and materials of the TX-TL cell-free expression system. Based on costs of reagents as of December 2012, and labor costs of $14 per hour. b) Comparison of TX-TL cell-free expression system costs vs. other commercial systems. Costs are broken down per &mu;l, although reaction volumes may vary per kit. c) Comparison of TX-TL cell-free expression system yield vs. other commercial systems. Protein expression yield determined by manufacturer standards. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig3highres.jpg" src="/files/ftp_upload/50762/50762fig3.jpg" /> 
Figure 3. Loading and processing of a bead-beating tube. a) Demonstration of correct viscosity of cell-bead solution. Cell-bead solution will have a viscosity dependent on many factors, including amount of S30A buffer added, amount of beads added, and time spent on ice. b) Loading of bead-beating tube before quick tabletop centrifugation. The centrifugation removes bubbles accumulated during loading. c) Bubbles surfacing after tabletop centrifugation. The size of the bubbles will vary; they can be popped or removed using a pipette tip. d) Completely filled bead-beating tube before capping. A meniscus is formed in the bead-beating tube, and the cap has enough to cover and cause small amounts to overfill. e) Correctly loaded filter apparatus. These can be reused. f) Comparison of correctly vs. incorrectly processed bead-beating tube. The tube on the left is a well-beat tube - it features a small and well-delineated top layer, and very clear supernatant. The tube on the right is suboptimal, based on the larger, hazy second layer and the hazy supernatant. Tubes that are suboptimal should not undergo additional processing.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig4highres.jpg" src="/files/ftp_upload/50762/50762fig4.jpg" /> 
Figure 4. Properties of crude extract preparations. a) Typical calibration plots for crude cell extract. Crude extract is calibrated for additional Mg-glutamate, K-glutamate, and DTT levels, in that order. Shown is endpoint fluorescence after 8 hr, as well as maximal rate of protein production based on a 12-minute moving average. Based on these plots, an acceptable range of additional Mg-glutamate is 4 mM, K-glutamate is 60-80 mM, and DTT is 0-3 mM. Note that every crude extract needs to be calibrated independently for these three variables. b) Variation from extract preparations. Endpoint fluorescence of two crude extracts prepared on different dates is shown; error bars are 1 standard deviation from three independent runs on different days. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig5highres.jpg" src="/files/ftp_upload/50762/50762fig5.jpg" /> 
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1 nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 1) or post-processed with a QiaQuick PCR purification kit (Purification method 2). Shown is endpoint fluorescence after 8 hr, as well as maximal rate of protein production based on a 12-minute moving average. Error bars are 1 standard deviation from four independent runs on different days. b) Effect of elution buffer (Tris-Cl). Different concentrations of Tris-Cl are compared in a cell-free expression reaction based on the expression of 1 nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500. Concentrations given are final concentrations of Tris-Cl in the reaction; elution buffer used is 10 mM Tris-Cl. Error bars are 1 standard deviation from three independent runs on different days. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig6highres.jpg" src="/files/ftp_upload/50762/50762fig6.jpg" /> 
Figure 6. Sample TX-TL run of a negative feedback loop. a) Sample setup of a cell-free execution reaction. Tests "on" vs. "off" state of the negative feedback loop, with positive and negative controls. b) Plasmid map of negative feedback loop. c) Representative results. Data reflects experiment in a) and b), with negative control subtracted from signal. Genetic circuit shown in insert. Error bars are 1 standard deviation from three independent runs on different days. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig6large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Concentration</td>
 <td>Amount</td>
 <td>Sterilization</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>Chloramphenicol (Cm)</td>
 <td>34 mg/ml in ethanol</td>
 <td>1 ml</td>
 <td>Filter sterilize (0.22 &mu;M)</td>
 <td>Can be made in larger volumes stored at -20 &deg;C for later use.</td>
 </tr>
 <tr>
 <td>2xYT+P+Cm agar plate</td>
 <td>31 g/L 2xYT, 40 mM potassium phosphate dibasic, 22 mM potassium phosphate monobasic, 34 &mu;g/ml chloramphenicol</td>
 <td>1 plate</td>
 <td>Autoclave</td>
 <td></td>
 </tr>
 <tr>
 <td>2xYT+P media</td>
 <td>31 g/L 2xYT, 40 mM potassium phosphate dibasic, 22 mM potassium phosphate monobasic </td>
 <td>4 L</td>
 <td>Autoclave</td>
 <td></td>
 </tr>
</tbody>
</table>
Table 1. Reagents for day 1 of Crude Cell Extract protocol.
<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Concentration</td>
 <td>Amount</td>
 <td>Sterilization</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>Tris base</td>
 <td>2 M</td>
 <td>250 ml</td>
 <td>Filter sterilize (0.22 &mu;M) or autoclave</td>
 <td>Can be stored at room temperature.</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>1 M</td>
 <td>6 ml</td>
 <td>Filter sterilize (0.22 &mu;M)</td>
 <td>Can be made in larger volumes and stored at -20 &deg;C for later use.</td>
 </tr>
 <tr>
 <td>S30A buffer</td>
 <td>14 mM Mg-glutamate, 60 mM K-glutamate, 50 mM Tris, pH 7.7</td>
 <td>2 L</td>
 <td>Autoclave</td>
 <td>To reach pH 7.7, titrate with acetic acid. Add DTT to 2 mM final concentration just before use. Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>S30B buffer</td>
 <td>14 mM Mg-glutamate, 60 mM K-glutamate, ~5 mM Tris, pH 8.2</td>
 <td>2 L</td>
 <td>Autoclave</td>
 <td>To reach pH 8.2, titrate with 2M Tris. Add DTT to 1 mM final concentration just before use. Store at 4 &deg;C.</td>
 </tr>
</tbody>
</table>
Table 2. Reagents for day 2 of Crude Cell Extract protocol.
<table border="1">
<tbody>
 <tr>
 <td></td>
 <td colspan="4">Falcon</td>
 </tr>
 <tr>
 <td></td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 </tr>
 <tr>
 <td>Empty 50 ml Falcon (g)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml Falcon with pellet (g)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pellet mass (50 ml Falcon with pellet - empty 50 ml Falcon) (g)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>S30A buffer volume to add (pellet mass* 0.9) (ml)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Total mass of beads to add (pellet mass * 5.0) (g)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
</tbody>
</table>
Table 3. S30A buffer and bead mass calculator, for day 3 of Crude Cell Extract protocol.
<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Concentration</td>
 <td>Amount</td>
 <td>Sterilization</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>2 M, pH 8</td>
 <td>4 ml</td>
 <td>None</td>
 <td>To reach pH 8, titrate with KOH.</td>
 </tr>
 <tr>
 <td>Nucleotide Mix</td>
 <td>156 mM ATP and GTP, 94 mM CTP and UTP, pH 7.5</td>
 <td>1.5 ml</td>
 <td>None</td>
 <td>To reach pH 7.5, titrate with KOH. </td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>50 mg/ml</td>
 <td>600 &mu;l</td>
 <td>None</td>
 <td></td>
 </tr>
 <tr>
 <td>CoA</td>
 <td>65 mM</td>
 <td>600 &mu;l</td>
 <td>None</td>
 <td></td>
 </tr>
 <tr>
 <td>NAD</td>
 <td>175 mM, pH 7.5-8</td>
 <td>300 &mu;l</td>
 <td>None</td>
 <td>To reach pH 7.5-8, titrate with Tris at 2 M.</td>
 </tr>
 <tr>
 <td>cAMP</td>
 <td>650 mM, pH 8</td>
 <td>200 &mu;l</td>
 <td>None</td>
 <td>To reach pH 8, titrate with Tris at 2 M.</td>
 </tr>
 <tr>
 <td>Folinic Acid</td>
 <td>33.9 mM</td>
 <td>300 &mu;l</td>
 <td>None</td>
 <td>Although only 300 &mu;l is needed, recipe in supplemental is for 1.15 ml.</td>
 </tr>
 <tr>
 <td>Spermidine</td>
 <td>1 M</td>
 <td>150 &mu;l</td>
 <td>None</td>
 <td>Store at 4 &deg;C, heat to 37 &deg;C to melt.</td>
 </tr>
 <tr>
 <td>3-PGA</td>
 <td>1.4 M, pH 7.5</td>
 <td>3.2 ml</td>
 <td>None</td>
 <td>To reach pH 7.5, titrate with Tris at 2 M.</td>
 </tr>
</tbody>
</table>
Table 4. Reagents to prepare for Energy Solution protocol.
 Supplemental Material 1. Recipes for Items. 
Chloramphenicol, 34 mg/ml: Prepare 0.51 g chloramphenicol and add ethanol to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use.
 2xYT+P+Cm agar plate: Prepare 1.24 g 2xYT, 1.6 ml potassium phosphate dibasic solution @ 1 M, 0.88 ml potassium phosphate monobasic solution @ 1 M, 0.6 g agar, and water to 40 ml. Autoclave. Let cool to 50 &deg;C and add 40 &mu;l Cm. Aliquot 25 ml into a 100 x 15 mm Petri dish, and let cool for an hour.
 2xYT+P media: Prepare 124 g 2xYT, 160 ml potassium phosphate dibasic solution @1 M, 88 ml potassium phosphate monobasic solution @ 1 M, and water to 4 L. Aliquot out into 2 x 1.88 L and 0.24 L. Autoclave.
 Tris base, 2 M: Prepare 60.57 g Tris base and water to 250 ml. Sterilize, store at RT for later use.
 DTT, 1 M: Prepare 2.31 g DTT and water to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use.
 S30A buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, 50 ml Tris at 2M, acetic acid (to pH 7.7), and water to 2 L. Autoclave, store at 4 &deg;C, add 4 ml 1 M DTT before use.
 S30B buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, Tris at 2 M (to pH 8.2), and water to 2 L. Autoclave, store at 4 &deg;C, add 2 ml 1 M DTT before use.
 HEPES: Prepare 1.91 g HEPES (MW 238.21), KOH (to pH 8), and water to 4 ml.
 tRNA: Prepare 30 mg of tRNA and water to 600 &mu;l.
 CoA: Prepare 30 mg of CoA (MW 767.53) and water to 600 &mu;l.
 NAD: Add 34.83 mg of NAD (MW 663.43), Tris at 2 M (to pH 7.5-8), and water to 300 &mu;l. (Add 27 &mu;l of Tris at 2 M to bring the solution to pH 7.5-8).
 cAMP: Add 42.80 mg of cAMP (MW 329.22), Tris at 2 M (to pH 8), and water to 200 &mu;l. (Add 73 &mu;l of Tris at 2 M to bring the solution to pH 8).
 Folinic Acid (33.9 mM): To 20 mg of solid folinic acid calcium salt (MW 511.5), add 1.15 ml water.
 Spermidine: Prepare 23.55 &mu;l of spermidine (MW 145.25) and water to 150 &mu;l. Prepare at room temperature after melting briefly at 37 &deg;C. 
 3-PGA: Add 1.03 g of 3-PGA (MW 230.02), Tris at 2 M (to pH 7.5), and water to 3.2 ml. (Add 1.73 ml of Tris at 2 M to bring the solution to pH 7.5).
Nucleotide Mix: Add 145 mg of ATP dipotassium salt dihydrate (MW 619.4), 133 mg of GTP disodium salt (MW 567.14), 79.4 mg of CTP disodium salt dihydrate (MW 563.16), 82.6 mg of UTP trisodium salt dihydrate (MW 586.12), KOH at 15% dilution (to pH 7.5), and water to 1.5 ml. (Add 353 &mu;l of KOH at 15% dilution to bring the solution to pH 7.5).
Supplemental Material 2. Bradford Assay.
<ol>
<li>Remove Bradford agent from 4 &deg;C and set at room temperature.</li>
<li>Prepare 50 &mu;l BSA Standard at 1 mg/ml and at 0.1 mg/ml.</li>
<li>Prepare 40 &mu;l 20x dilution of extract from step 1.47.</li>
<li>Add 800 &mu;l water to 7 cuvettes. </li>
<li>Prepare standard cuvettes for 0 mg/ml, 1 mg/ml (10 &mu;l 0.1 mg/ml BSA), 2 mg/ml (20 &mu;l 0.1 mg/ml BSA), 4 mg/ml (4 &mu;l 1 mg/ml BSA), 6 mg/ml (6 &mu;l 1 mg/ml BSA).</li>
<li>Prepare experimental cuvettes for 2 &mu;l of sample and 4 &mu;l of sample.</li>
<li>Add 200 &mu;l of Bradford agent to each cuvette and mix well by pipetting. Incubate at room temperature for at least 10 min.</li>
<li>Produce standard curve at OD 595 nm using cuvettes from step 6.5. Reject standard curve if r2 &lt; 0.95.</li>
<li> Determine extract concentration at OD 595 nm using cuvettes from step 6.6.</li>
</ol>
Supplemental Material 3. Buffer calibration spreadsheet.
<a href="https://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank"> See TXTL_e(template)_calibration_JoVE.xlsx</a>.
Supplemental Material 4. Cell-free expression run spreadsheet.
<a href="https://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">See TXTL _JoVE.xlsx</a>.

Watch this Scientific Journal Video about Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology at JoVE.com

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

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JoVE Journal

Biology

62.2K vistas.  California Institute of Technology.  Este protocolo de cinco días se esbozan todos los pasos, el equipo y el software complementario necesario para la creación y ejecución de un eficiente Escherichia coli endógeno basado sistema de expresión libre de células TX-TL a partir de cero. Con los reactivos, el protocolo de toma 8 horas o menos a la configuración de una reacción, recopilar y procesar datos. 