ACB and JLF acknowledge funding support by the ANR SINAPUV grant (ANR-17-CE07-0046). JLF and JB acknowledge funding support by the ANR SynBioDiag grant (ANR-18-CE33-0015). MSA and JLF acknowledge funding support by the ANR iCFree grant (ANR-20-BiopNSE). JB acknowledges support from the ERC starting &#34;COMPUCELL&#34; (grant number 657579). The Centre de Biochimie Structurale acknowledges support from&#160;the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INSB-05-01). MK acknowledges funding support from INRAe&#39;s MICA department, Universit&#233; Paris-Saclay, Ile-de-France (IdF) region&#39;s DIM-RFSI, and ANR DREAMY (ANR-21-CE48-003). This work was supported by funding through the European Research Council Consolidator Award (865973 to CLB).

1. Media and buffer preparation
<ol>
	<li>Preparation of 2xYT+P medium (solid and liquid)
	<ol>
		<li>Prepare liquid and solid media as described in Table 1, and then sterilize by autoclaving. 
		NOTE: This protocol requires one 2xYT+P agar plate containing chloramphenicol (10 &#181;g/mL). The volume of liquid media is proportional to the volume of the lysate required. Here, a starting volume of 5 L of liquid 2xYT+P media is used. However, the volume can be adjusted as needed, kno...

<ol>
	<li>Silverman, A. D., Karim, A. S., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+gene+expression:+an+expanded+repertoire+of+applications.">Cell-free gene expression: an expanded repertoire of applications.</a> Nature Reviews Genetics. 21 (3), 151-170 (2020).</li><li>McSweeney, M. A., Styczynski, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+use+of+linear+DNA+in+cell-free+expression+systems.">Effective use of linear DNA in cell-free expression systems.</a> Frontiers in Bioengineering and Biotechnology. 9, 715328 (2021).</li><li>Schinn, S. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+directly+from+PCR:+progress+and+applications+of+cell-free+protein+synthesis+with+linear+DNA.">Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.</a> New Biotechnology. 33 (4), 8 (2016).</li><li>Prell, A., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+linear+and+circular+DNA+with+gaps+by+the+recBC+enzyme+of+Escherichia+coli.+Effects+of+gap+length+and+the+presence+of+cell-free+extracts.">Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli. Effects of gap length and the presence of cell-free extracts.</a> European Journal of Biochemistry. 105 (1), 109-116 (1980).</li><li>Sitaraman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cell-free+protein+synthesis+system.">A novel cell-free protein synthesis system.</a> Journal of Biotechnology. 110 (3), 257-263 (2004).</li><li>Marshall, R., Maxwell, C. S., Collins, S. P., Beisel, C. L., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+DNA+containing+&#967;+sites+enhances+DNA+stability+and+gene+expression+in+E.+coli+cell-free+transcription-translation+systems.">Short DNA containing &#967; sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.</a> Biotechnology and Bioengineering. 114 (9), 2137-2141 (2017).</li><li>Yim, S. S., Johns, N. I., Noireaux, V., Wang, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+linear+DNA+templates+in+cell-free+expression+systems+from+diverse+bacteria.">Protecting linear DNA templates in cell-free expression systems from diverse bacteria.</a> ACS Synthetic Biology. 9 (10), 2851-2855 (2020).</li><li>Norouzi, M., Panfilov, S., Pardee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+protection+of+linear+DNA+in+cell-free+extracts+from+Escherichia+coli+and+Vibrio+natriegens.">High-efficiency protection of linear DNA in cell-free extracts from Escherichia coli and Vibrio natriegens.</a> ACS Synthetic Biology. 10 (7), 1615-1624 (2021).</li><li>Zhu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+cell-free+gene+expression+yields+from+linear+templates+in+Escherichia+coli+and+Vibrio+natriegens+extracts+by+using+DNA-binding+proteins.">Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins.</a> Biotechnology and Bioengineering. 117 (12), 3849-3857 (2020).</li><li>Batista, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+optimized+cell-free+buffer+enables+robust+expression+from+unprotected+linear+DNA+in+exonuclease-deficient+extracts.">Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts.</a> ACS Synthetic Biology. 11 (2), 732-746 (2022).</li><li>Levine, M. Z., Gregorio, N. E., Jewett, M. C., Watts, K. R., Oza, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli-based+cell-free+protein+synthesis:+protocols+for+a+robust,+flexible,+and+accessible+platform+technology.">Escherichia coli-based cell-free protein synthesis: protocols for a robust, flexible, and accessible platform technology.</a> JoVE. (144), e58882 (2019).</li><li>Kwon, Y. -. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+preparation+methods+of+crude+extract+for+robust+cell-free+protein+synthesis.">High-throughput preparation methods of crude extract for robust cell-free protein synthesis.</a> Scientific Reports. 5 (1), 8663 (2015).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> Journal of Visualized Experiments. (79), e50762 (2013).</li><li>Dopp, J. L., Jo, Y. R., Reuel, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+reduce+variability+in+E.+Coli-based+cell-free+protein+expression+experiments.">Methods to reduce variability in E. Coli-based cell-free protein expression experiments.</a> Synthetic and Systems Biotechnology. 4 (4), 204-211 (2019).</li><li>Cole, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+interlaboratory+cell-free+protein+synthesis+variability.">Quantification of interlaboratory cell-free protein synthesis variability.</a> ACS Synthetic Biology. 8 (9), 2080-2091 (2019).</li></ol>

Here, we show that cell lysate prepared from E. coli BL21 Rosetta2 with a genomic knockout for either recB or recBCD operon supports high protein expression from linear DNA templates. This protocol elaborates a step-by-step lysate calibration procedure specific for linear DNA templates (Figure 2), which is a critical step that leads to the high expression from &#963;70 promoters in linear DNA, reaching near-plasmid levels for equimolar DNA concentrations. This prot...

Cell-free protein synthesis (CFPS) systems are increasingly being used as a fast, simple, and efficient method for biosensor engineering, decentralized manufacturing, and prototyping of genetic circuits1. Due to their great potential, CFPS systems are used regularly in the field of synthetic biology. However, so far CFPS systems rely on circular plasmids that can limit the technology from reaching its full potential. Preparing plasmid DNA depends on many time-consuming steps during&#160;cloning and&#160;large amounts of DNA isolation. On the other hand, PCR amplification from a plasmid, or a synthesized DNA template, can be used to simply prepare CFPS templates within a few hours2,3. Therefore, application of linear DNA offers a promising solution for CFPS. However, linear DNA is rapidly degraded by exonucleases naturally present in cellular extracts4. There are solutions that address this problem, such as using the &#955;-phage GamS protein5 or DNA containing Chi sites6 as protective agents, or directly protecting the linear DNA by chemical modification of its ends2,7,8,9. All these methods require supplementations to the cell extract, which are costly and time-consuming. It has been known for a long time that the exonuclease V complex (RecBCD) degrades linear DNA in cell lysates4. Recently, we showed that linear DNA can be much better protected in lysates from cells knocked out for exonuclease genes (recBCD)10.
In this protocol, steps for the preparation of cell-free lysates from the E. coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis are described in detail. Sonication lysis is a common and affordable technique employed by several labs11,12. The extracts produced from this strain do not need the addition of any extra component or DNA template modification to support expression from linear DNA templates. The method relies on the essential step of buffer optimization for cell extracts specifically for linear DNA expression from native E. coli promoters. It has been shown that this specific buffer optimization for linear DNA expression is the key for native &#963;70 promoters to yield high protein production without GamS protein or Chi DNA supplementation, even avoiding purification of the PCR products10. The optimal concentration of Mg-glu for linear DNA expression was found to be similar to that for plasmid DNA. However, the optimal concentration of K-glu showed a substantial difference between linear and plasmid DNA, likely due to a transcription-related mechanism10. The functionality of proteins expressed using this method has been demonstrated for several applications, such as rapid screening of toehold switches and activity assessment of enzyme variants10.
This protocol provides a simple, efficient, and cost-effective solution for using linear DNA templates in E. coli cell-free systems by simply using mutant &#916;recBCD cell extracts and specific calibration for linear DNA as template.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >2-YT Broth</td>
			<td >Invitrogen</td>
			<td >22712020</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL Safe-Lock tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL microtube</td>
		</tr>
		<tr >
			<td >384-well square-bottom microplate</td>
			<td>Thermo Scientific Nunc</td>
			<td>142761</td>
			<td ></td>
		</tr>
		<tr >
			<td >3PGA (D-(-)-3-Phosphoglyceric acid disodium)</td>
			<td>Sigma-Aldrich</td>
			<td>P8877</td>
			<td></td>
		</tr>
		<tr >
			<td >adhesive plate seal</td>
			<td>Thermo Scientific Nunc</td>
			<td>232701</td>
			<td ></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Invitrogen</td>
			<td>30391023</td>
			<td></td>
		</tr>
		<tr >
			<td >ATP (Adenosine 5&#39;-triphosphate disodium salt hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr >
			<td >BL21 Rosetta2</td>
			<td>Merck Millipore</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr >
			<td >BL21 Rosetta2 &#916;recB</td>
			<td>Addgene</td>
			<td>176582</td>
			<td></td>
		</tr>
		<tr >
			<td >BL21 Rosetta2 &#916;recBCD</td>
			<td>Addgene</td>
			<td>176583</td>
			<td></td>
		</tr>
		<tr >
			<td >cAMP (Adenosine 3&#39; 5&#39;-cyclic monophosphate)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr >
			<td >Chloramphenicol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr >
			<td >CoA (Coenzyme A hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>C4282</td>
			<td></td>
		</tr>
		<tr >
			<td >Corning 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar Cap, Sterile</td>
			<td>Corning</td>
			<td>430790</td>
			<td>15 mL tube</td>
		</tr>
		<tr >
			<td >Corning 50 mL PP Centrifuge Tubes, Conical Bottom with CentriStar Cap, Sterile</td>
			<td>Corning</td>
			<td>430828</td>
			<td>50 mL tube</td>
		</tr>
		<tr >
			<td >CTP (Cytidine 5&#39;-triphosphate, disodium salt hydrate)</td>
			<td>Alfa Aesar</td>
			<td>&#160;J62238</td>
			<td></td>
		</tr>
		<tr >
			<td >DpnI</td>
			<td>NEB</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr >
			<td >DTT (DL-Dithiothreitol)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr >
			<td >Folinic acid (solid folinic acid calcium salt)</td>
			<td>Sigma-Aldrich</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr >
			<td >GTP (Guanosine 5&#697;-Triphosphate,&#160; Disodium Salt)</td>
			<td>Sigma-Aldrich</td>
			<td>371701</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;H3375</td>
			<td></td>
		</tr>
		<tr >
			<td >K phosphate dibasic (K2HPO4)</td>
			<td>Carl Roth</td>
			<td>231-834-5</td>
			<td></td>
		</tr>
		<tr >
			<td >K phosphate monobasic (H2KO4P)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >K-glutamate</td>
			<td>Alfa Aesar</td>
			<td>A17232</td>
			<td></td>
		</tr>
		<tr >
			<td >Mg-glutamate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr >
			<td >Millex-GP Syringe Filter Unit, 0.22 &#181;m, polyethersulfone</td>
			<td>Merck Millipore</td>
			<td>SLGP033RB</td>
			<td>membrane filter 0.22 &#181;m</td>
		</tr>
		<tr >
			<td >Monarch PCR &#38; DNA Cleanup Kit</td>
			<td>NEB</td>
			<td>T1030S</td>
			<td>PCR &#38; DNA cleanup Kit</td>
		</tr>
		<tr >
			<td >Monarch Plasmid Miniprep Kit</td>
			<td>NEB</td>
			<td>T1010S</td>
			<td>Plasmid Miniprep Kit</td>
		</tr>
		<tr >
			<td >NAD (B-nicotinamide adenine dinucleotide hydrate)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>N6522</td>
			<td></td>
		</tr>
		<tr >
			<td >NIST-traceable FITC standard</td>
			<td>Invitrogen</td>
			<td>F36915</td>
			<td>NIST-FITC</td>
		</tr>
		<tr >
			<td >NucleoBond Xtra Maxi kit</td>
			<td>Macherey-Nagel</td>
			<td>740414.1</td>
			<td>Plasmid Maxiprep Kit</td>
		</tr>
		<tr >
			<td >PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr >
			<td >plate reader</td>
			<td>Biotek&#160;</td>
			<td>Synergy HTX</td>
			<td ></td>
		</tr>
		<tr >
			<td >Purified Recombinant EGFP Protein</td>
			<td>Chromotek</td>
			<td>egfp-250</td>
			<td>recombinant eGFP</td>
		</tr>
		<tr >
			<td >Q5 High-Fidelity 2X Master Mix</td>
			<td>NEB</td>
			<td>M0492S</td>
			<td>DNA polymerase</td>
		</tr>
		<tr >
			<td >Q5 High-Fidelity 2X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0492L</td>
			<td>DNA polymerase</td>
		</tr>
		<tr >
			<td >Qubit dsDNA BR Assay Kit</td>
			<td>Thermo</td>
			<td>Q32850</td>
			<td>fluorometric assay with DNA-binding dye</td>
		</tr>
		<tr >
			<td >RTS Amino Acid Sampler</td>
			<td>biotechrabbit</td>
			<td>BR1401801</td>
			<td></td>
		</tr>
		<tr >
			<td >Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>85558</td>
			<td></td>
		</tr>
		<tr >
			<td >Sterile water</td>
			<td></td>
			<td></td>
			<td >Purified water from the Millipore RiOs 8 system, sterilized by autoclaving.</td>
		</tr>
		<tr >
			<td >Tris base</td>
			<td>Life Science products Cytiva</td>
			<td>17-1321-01&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >tRNA</td>
			<td>Roche</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr >
			<td >Ultrasonic processor Vibra cell VC-505</td>
			<td>SONICS</td>
			<td>VC505</td>
			<td>sonicator</td>
		</tr>
		<tr >
			<td >UTP Na3 (Uridine 5&#39;- triphosphate, trisodium salt hydrate)</td>
			<td>Acros Organics&#160;</td>
			<td>226310010</td>
			<td></td>
		</tr>
		<tr >
			<td >Water, nuclease-free</td>
			<td>Thermo</td>
			<td>R0581</td>
			<td>nuclease-free water</td>
		</tr>
	</tbody>
</table>

cell-free protein synthesis from exonuclease-deficient cellular extracts utilizing linear dna templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Representative results are shown here after calibration of the lysate for optimal Mg-glutamate and K-glutamate levels separately for linear and plasmid DNA (Figure 1). The Mg-glutamate optimal concentration is similar across &#916;recB and &#916;recBCD extracts at 8 mM (Figure 1B). However, the optimal K-glutamate concentration for plasmid DNA is 140&#160;mM, whereas the optimal K-glutamate concentration for linear DNA for the same extract is 2...

Watch this Scientific Journal Video about Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates at JoVE.com

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...

The protocol is very significant because it enables fast, easy, and direct use of linear DNA templates in cell-free protein synthesis systems, even from native E.coli parameters. The main advantages are the time amount saved by avoiding cloning, chemical modifications of linear DNA ends and protective supplements like gamma or chi DNA. As self-reporting synthesis extracts are being developed for different organisms of interest, our method presents a way of speeding up prototyping in those extracts by using linear DNA.To begin, streak strain BL21 Rosetta-2 delta recBCD from minus 80 degrees Celsius glycerol stock onto a 2x YTP agar plate that contains 10 micrograms per milliliter chloramphenicol and incubate overnight at 37 degrees Celsius. Then, inoculate a single colony from the agar plate into a little more than 40 milliliters of 2x YTP supplemented with 10 micrograms per milliliter chloramphenicol and incubate overnight at 37 degree Celsius with 200 RPM shaking. Next, make a subculture by diluting the overnight culture 100 times into four liters of fresh 2x YTP media containing 10 micrograms per milliliter chloramphenicol.Divide the four liters of the media into four separate flasks. Incubate at 37 degrees Celsius, 200 RPM, for about three to four hours of growth to reach an optical density of 1.5 to 2.0. Put the culture on ice, spin down the cells at 5, 000 G for 12 minutes at four degree Celsius, and then discard the supernatant by decanting.Then, resuspend the cell pellets in 800 milliliters of chilled S30A with DTT. Again, spin down the cells at 5, 000 G for 12 minutes at four degree Celsius as demonstrated previously and discard the supernatant by decanting. After resuspending cell pellets in 160 milliliters of chilled S30A with DTT, transfer the cells to chilled and pre-weighed 50-milliliter tubes.Spin down the cells at 2000 G for eight minutes at four degree Celsius and then discard the supernatant by decanting. Again, spin down the cells at 2000 G for four minutes at four degrees Celsius, and then remove the remaining supernatant carefully by using a pipette. Then, remeasure the weight of the tube to calculate the weight of the cell pellet and keep the cell pellets at minus 80 degrees Celsius.After taking the cell pellets from minus 80 degrees Celsius, thaw them on ice for one to two hours and resuspend the cell pellets in 0.9 milliliters of S30A with DTT buffer per gram of the cell pellet's weight. Then, pipette slowly to resuspend the cells, avoiding top froth as much as possible. Place one-milliliter aliquots of the resuspended cells into 1.5-milliliter microtubes and keep them on ice or a pre-chilled cold block at four degree Celsius.Next, sonicate each tube in a sonicator with a setup of 20%amplitude for three cycles and spin down the lysate at 12, 000 G for 10 minutes at four degree Celsius. After collecting the supernatant with a pipette, transfer to a 50-milliliter tube. Incubate the cell lysate at 37 degrees Celsius at 200 RPM agitation for 80 minutes.After spinning down the lysate and collecting the supernatant in pre-chilled 1.5-milliliter microtubes, aliquot 30 microliters in pre-chilled PCR 8-strip tubes, while keeping all tubes on ice. Flash freeze the lysate aliquots on dry ice and store at minus 80 degrees Celsius. Prepare a master mix according to the buffer preparation tab as described in the manuscript.For a reaction volume of 10.5 microliters, add 1.05-microliter of different Mg-glutamate stock concentrations and 9.45 microliters of the master mix and mix gently. Pipette 10 microliters of the above reactions into a 384-well square bottom microplate and cover it using an adhesive plate seal. Measure gene expression as fluorescence output by recording fluorescence data with a plate reader at regular intervals for eight hours of incubation at 30 degrees Celsius with continuous orbital shaking at 307 cycles per minute.After identifying the Mg-glutamate concentration that results in the highest fluorescence value at the end point, proceed to K-glutamate calibration. Run the experiment and identify the highest fluorescence value from among the K-glutamate concentrations tested, thus identifying the optimal buffer composition for Mg-glutamate and K-glutamate for this lysate. Once the Mg-glutamate and K-glutamate values are established, prepare stock tubes of the optimized buffer composition according to the batch volume obtained.After calculating the number of buffer tubes needed, aliquot 38 microliters per tube and store at minus 80 degrees Celsius. First, label a 1.5-milliliter microtube for the optimal master mix preparation. Add the correct volumes of the buffer and lysate and mix them gently.Then, pipette the DNA samples first, followed by nuclease-free water, and finally, the optimal master mix. Mix the cell-free reaction gently with a pipette just before adding to the plate reader and avoid any bubbles. After setting up the reactions in the plate reader, kinetic runs recorded fluorescence data at regular intervals for eight hours of incubation at 30 degrees Celsius with continuous orbital shaking at 307 cycles per minute.After calibration of the lysate, the Mg-glutamate optimal concentration was similar at eight millimolar across extracts for both linear and plasmid DNA. However, the optimal K-glutamate concentration for plasmid DNA is 140 millimolar, whereas the optimal K-glutamate concentration for linear DNA is 20 millimolar. After calibration steps, levels of GFP expressions were compared between linear and plasmid DNA in the wild-type, delta recB, and delta recBCD extracts.The GFP expression from linear DNA reached 102%and 138%of the expression from plasmid DNA in extracts from delta recB and delta recBCD strains, respectively. The most important things in the protocol are sonication lysis of the cell and the buffer calibration steps separately for linear and plasmid DNA, particularly when using the native parameters. This technique would help speed up the testing of biological designs in cell-free systems, for example, the screening of biosensors, enzymes, and pathways, or prototyping of synthetic genetic circuits.

cell-free-protein-synthesis-from-exonuclease-deficient-cellular

Research

JoVE Journal

Bioengineering

3.3K visualizações.  Université Paris-Saclay. O protocolo é muito significativo porque permite o uso rápido, fácil e direto de modelos lineares de DNA em sistemas de síntese de proteínas livres de células, mesmo a partir de parâmetros nativos de E.coli. As principais vantagens são a quantidade de tempo economizada ao evitar a clonagem, modificações químicas de extremidades lineares de DNA e suplementos protetores como DNA gama ou chi. Como os extratos de síntese de autorrelato estão sendo desenvolvidos para diferentes organi...