Name: protocols for implementing an escherichia coli based tx-tl cell-free expression system for synthetic biology
Uploaded: 2013-09-16T13:00:00
Duration: 16 min 11 s
Description: 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

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.

<ol>
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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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...