Name: cell-free protein synthesis from exonuclease-deficient cellular extracts utilizing linear dna templates
Uploaded: 2022-08-09T14:00:00
Description: Watch this Scientific Journal Video about Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates at JoVE.com

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. -. 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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

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

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

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

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

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

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

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

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

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

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

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

OnePot PURE Cell-Free System

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

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

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

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

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

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

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