The authors thank Zachary Sun and Richard Murray (California Institute of Technology) for kindly providing the plasmid P_araBAD-gamS, and Kaeko Kamei (Kyoto Institute of Technology) for kindly providing a high-speed cooling centrifuge. This work was supported by grants from the National Institutes of Health and from the ARO MURI program and was partly supported by the Leading Initiative for Excellent Young Researchers, MEXT, Japan.

1. Prepare media and buffers.
<ol>
	<li>Prepare 2xYTPG medium.
	<ol>
		<li>Mix 62 g 2xYT powder, 5.99 g potassium phosphate monobasic, 13.93 g potassium phosphate dibasic, and deionized water to 2 L.</li>
		<li>Autoclave on liquid cycle with an exposure time of 30 min14.</li>
		<li>To 400 mL of 2xYTP media from 1.1.2, add 7.2 g D-glucose (dextrose) and mix until dissolved.</li>
		<li>Filter-sterilize through a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Prepare S30A buffer.
	<ol>
		<li>Mix Tris-HCl (pH 7.7, 50 mM final concentration), potassium glutamate (60 mM final), and magnesium glutamate (14 mM final).</li>
		<li>Adjust the pH to 7.7 using 10 M KOH.</li>
	</ol>
	</li>
	<li>Prepare Solution 1 (see Table 1).
	<ol>
		<li>Resuspend 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) in 2 mL of water.</li>
		<li>Adjust the pH to 8.0 using KOH.</li>
		<li>Add all other components from Table 1.</li>
		<li>Adjust the pH to 7.6 using 10 M KOH. Filter-sterilize.</li>
	</ol>
	</li>
	<li>Prepare 2.5x premix solution (see Table 2).
	<ol>
		<li>Mix all the components in Table 2.</li>
		<li>Adjust the pH to 7.5 using KOH.</li>
		<li>Aliquot and freeze at -80 &#176;C. 
		&#8203;NOTE: A 20 &#181;L reaction uses 8.9 &#181;L of premix.</li>
	</ol>
	</li>
</ol>
2. Prepare cells.
<ol>
	<li>Streak the autolysate cells onto LB agar plates containing 50 &#181;g/mL ampicillin using an inoculating loop and grow at 37 &#176;C (see Note 1).</li>
	<li>Pick a single colony into a starter culture of LB/ampicillin medium using a pipet tip and grow at 37 &#176;C overnight.</li>
	<li>Inoculate 400 mL of 2xYTPG medium containing 50 &#181;g/mL ampicillin with 400 &#181;L of starter culture, and grow at 37 &#176;C in a 1 L Erlenmeyer flask, shaking at 300 rpm.</li>
	<li>Periodically measure the culture&#39;s optical density at 600 nm (OD600) using a spectrophotometer to read an optical cuvette with a 1 cm path length. When the OD600 exceeds 1, begin diluting the culture 5-fold before measurements to ensure that the measurements remain within the linear range of a typical laboratory spectrophotometer. Continue growing the cells until the 5-fold diluted culture reaches OD600 of 0.3 (corresponding to a culture OD600 of 1.5).</li>
</ol>
3. Prepare the lysate.
<ol>
	<li>Prepare S30A buffer supplemented with 2 mM dithiothreitol (DTT). Mix 3&#160;mL of S30A buffer with 6&#160;&#181;L of DTT stock solution at 1 M. Place on ice for use in step 3.7.</li>
	<li>Harvest the cells by centrifuging at 1800 &#215; g for 15 min at room temperature.</li>
	<li>Discard the supernatant by pouring it off and using a pipet to remove any remaining liquid.</li>
	<li>Resuspend the pellet in 45 mL of cold (4-10 &#176;C) S30A buffer using a vortex mixer.</li>
	<li>Weigh an empty 50 mL centrifuge tube, transfer the cells into it, and repeat steps 3.2-3.3 to wash the cells.</li>
	<li>Weigh the pellet, subtracting the weight of an empty 50 mL tube. Make sure to carefully aspirate any remaining supernatant to ensure an accurate measurement of pellet weight. 
	NOTE: A typical yield is ~1.3 g of cell pellet from 400 mL of production culture.</li>
	<li>Add 2 volumes of cold S30A buffer supplemented with 2 mM dithiothreitol, i.e., 2 mL of buffer for every 1 g of cell pellet, and resuspend the cells by vigorously vortex mixing.</li>
	<li>Freeze the cells. Place the 50 mL tube containing the&#160;cells in a -20 &#176;C or -80 &#176;C freezer until the pellet is thoroughly frozen. 
	NOTE: The freezing step is a good stopping point for the day.</li>
	<li>Thaw the cells in a room temperature water bath.</li>
	<li>Vortex vigorously for 2-3 min.</li>
	<li>Incubate at 37 &#176;C for 45 min with shaking at 300 rpm.</li>
	<li>Clear the sample of heavy cellular debris by centrifuging in transparent centrifuge tubes at 30,000 &#215; g for 45 min at 4 &#176;C. 
	NOTE: If a centrifuge capable of 30,000 x g is not available, centrifuge for 45 min at 21,000 &#215; g, and use additional caution in step 3.13, as the pellet will be less compact.</li>
	<li>Carefully transfer the supernatant to a new tube with a pipet, avoiding disturbing the pellet as much as possible. If the transferred supernatant is contaminated with material from the pellet, repeat the previous step.</li>
	<li>Transfer the supernatant to 1.5 mL centrifuge tubes and centrifuge once more at 21,000 &#215; g (or the maximum speed of a tabletop centrifuge) for 5 min.</li>
	<li>Aliquot the cleared autolysate into the desired volumes, carefully avoiding any remaining pellet, and freeze at -80 &#176;C or use immediately. 
	&#8203;NOTE: A single 20 &#181;L reaction uses 8 &#181;L of autolysate.</li>
</ol>
4. Cell-free gene expression
NOTE: The autolysate is now ready for any desired end-use. The following is an example standard protocol for cell-free gene expression.
<ol>
	<li>For a 20 &#181;L reaction, mix on ice 8 &#181;L of autolysate and 8.9 &#181;L of premix. See&#160;NOTE&#160;at the end of the protocol section regarding the optimization of magnesium glutamate and PEG 8000 concentrations.</li>
	<li>Add DNA (e.g., pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 to a final concentration of 8 nM), any other reagents, and water to 20 &#181;L.</li>
	<li>Place the reaction in a 384-well microplate and measure the fluorescence time course and/or endpoints using a plate reader. For green fluorescent protein (GFP), use an excitation wavelength of 485 nm and an emission wavelength of 520 nm.</li>
</ol>
5. Protocol modifications
NOTE: The following modifications of the protocol enable it to serve other applications.
<ol>
	<li>Cell-free gene expression using linear DNA templates
	<ol>
		<li>Perform the steps in section 4, supplementing the reaction with 2.2 &#181;M purified GamS protein (expressed and purified as described12) before the addition of the linear DNA.</li>
	</ol>
	</li>
	<li>Cell-free gene expression incorporating protein degradation
	<ol>
		<li>In step 2.1, use autolysate cells containing the plasmid pACYC-FLAG-dN6-His (see the Table of Materials). In all growth media, additionally include 34 &#181;g/mL chloramphenicol.</li>
		<li>In step 2.3, include 40 &#181;M isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) in the growth medium to induce expression from the plasmid.</li>
		<li>Repeat steps 3.2-3.4 (washing) two additional times (for a total of three washes) to ensure the complete removal of chloramphenicol, which is a translation inhibitor. For the first two washes (step 3.4), substitute S30A buffer with phosphate-buffered saline (pH 7.4).</li>
		<li>In step 4.2, supplement with an additional 3 mM ATP (added from a stock solution of 100 mM ATP in water, pH 7.2) and 4.5 mM magnesium glutamate (using a 1 M stock solution in water) (final concentrations) to compensate for high ATP use by ClpXP, as well as chelation of magnesium by the additional ATP.</li>
	</ol>
	</li>
	<li>Cell-free gene expression using LacZ-based readouts (including colorimetric)
	<ol>
		<li>In step 2.1, use autolysate cells that do not natively express LacZ (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Alternatively, prepare the lysate directly from freeze-dried cells.
	<ol>
		<li>Perform all steps from 1.1 to 3.7.</li>
		<li>Mix 8 &#181;L of cell suspension with 8.9 &#181;L of premix.</li>
		<li>Add plasmid DNA (if desired), other custom reagents, and water to reach a final volume of 20 &#181;L.</li>
		<li>Transfer the reaction to a 384-well microplate and freeze-dry it. 
		NOTE: Freeze-dried samples can be stored for up to a week and possibly longer.</li>
		<li>To begin the reaction, rehydrate it with 18 &#181;L of deionized water supplemented with any desired DNA or other reagents.</li>
		<li>Follow the fluorescence dynamics in a plate reader.</li>
	</ol>
	</li>
</ol>
NOTE&#160;1: Autolysate cells are designed to lyse upon a freeze-thaw cycle, so it is particularly important to use cryoprotectant when making frozen stocks. We froze stocks in 24% wt/vol glycerol and stored them at -80 &#176;C.
NOTE 2: Magnesium ions and PEG 8000 are critical for lysate performance. The base 2.5x premix, based on previously published data, contains 6 mM magnesium glutamate and 4.8% wt/vol PEG 8000, which become 2.4 mM and 1.9%, respectively, in the final reaction. The autolysate prepared with the protocol here typically performs best with an additional 5 mM Mg-glutamate and 1.5% PEG 8000 in the final reaction. However, this can be optimized in the range of an additional 0-10 mM Mg glutamate and an additional 0-3% PEG 8000 (compared to the base premix). To prepare the premix with the recommended additional 5 mM Mg-glutamate and 1.5% PEG 8000 (final concentrations), mix 380 &#181;L of premix with 4.75 &#181;L of magnesium glutamate at 1 M and 36.1 &#181;L of PEG 8000 at 40% weight/volume.

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	<li>Dudley, Q. M., 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+metabolic+engineering:+biomanufacturing+beyond+the+cell.">Cell-free metabolic engineering: biomanufacturing beyond the cell.</a> Biotechnology Journal. 10 (1), 69-82 (2015).</li><li>Smith, M. T., Wilding, K. M., Hunt, J. M., Bennett, A. M., Bundy, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+age+of+cell-free+synthetic+biology.">The emerging age of cell-free synthetic biology.</a> FEBS Letters. 588 (17), 2755-2761 (2014).</li><li>Casteleijn, M. G., Urtti, A., Sarkhel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+without+boundaries:+cell-free+protein+synthesis+in+pharmaceutical+research.">Expression without boundaries: cell-free protein synthesis in pharmaceutical research.</a> International Journal of Pharmaceutics. 440 (1), 39-47 (2013).</li><li>He, M. <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+protein+synthesis:+applications+in+proteomics+and+biotechnology.">Cell-free protein synthesis: applications in proteomics and biotechnology.</a> New Biotechnology. 25 (2-3), 126-132 (2008).</li><li>Pardee, 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=Paper-based+synthetic+gene+networks.">Paper-based synthetic gene networks.</a> Cell. 159 (4), 940-954 (2014).</li><li>Pardee, 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=low-cost+detection+of+Zika+virus+using+programmable+biomolecular+components.">low-cost detection of Zika virus using programmable biomolecular components.</a> Cell. 165 (5), 1255-1266 (2016).</li><li>Takahashi, M. 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=Characterizing+and+prototyping+genetic+networks+with+cell-free+transcription-translation+reactions.">Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.</a> Methods. 86, 60-72 (2015).</li><li>Siegal-Gaskins, D., Tuza, Z. A., Kim, J., Noireaux, V., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+circuit+performance+characterization+and+resource+usage+in+a+cell-free+"breadboard".">Gene circuit performance characterization and resource usage in a cell-free "breadboard".</a> ACS Synthetic Biology. 3 (6), 416-425 (2014).</li><li>Niederholtmeyer, H., Chaggan, C., Devaraj, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+and+quorum+sensing+in+non-living+mimics+of+eukaryotic+cells.">Communication and quorum sensing in non-living mimics of eukaryotic cells.</a> Nature Communications. 9, 5027 (2018).</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: JoVE. (79), e50762 (2013).</li><li>Dopp, J., Jo, Y., Reuel, N. <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+systems.">Methods to reduce variability in E. coli-based cell-free protein expression systems.</a> Synthetic and Systems Biotechnology. 4 (4), 204-211 (2019).</li><li>Didovyk, A., Tonooka, T., Tsimring, L., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+scalable+preparation+of+bacterial+lysates+for+cell-free+gene+expression.">Rapid and scalable preparation of bacterial lysates for cell-free gene expression.</a> ACS Synthetic Biology. 6 (12), 2198-2208 (2017).</li><li>Tonooka, T., Niederholtmeyer, H., Tsimring, L., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cell+on+a+chip+integrated+with+protein+degradation.">Artificial cell on a chip integrated with protein degradation.</a> 2019 IEEE 32nd International Conference on Micro Electro Mechanical Systems (MEMS). , 107-109 (2019).</li><li>Garibaldi, 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=Validation+of+autoclave+protocols+for+successful+decontamination+of+category+A+medical+waste+generated+from+care+of+patients+with+serious+communicable+diseases.">Validation of autoclave protocols for successful decontamination of category A medical waste generated from care of patients with serious communicable diseases.</a> Journal of Clinical Microbiology. 55 (2), 545-551 (2017).</li><li>Cole, S., Miklos, A., Chiao, A., Sun, Z., Lux, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+preparation+of+prokaryotic+extracts+for+cell-free+expression+systems.">Methodologies for preparation of prokaryotic extracts for cell-free expression systems.</a> Synthetic and Systems Biotechnology. 5 (4), 252-267 (2020).</li><li>Fujiwara, K., Doi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+preparation+of+cell+extract+for+cell-free+protein+synthesis+without+physical+disruption.">Biochemical preparation of cell extract for cell-free protein synthesis without physical disruption.</a> PLoS ONE. 11 (4), 0154614 (2016).</li><li>Shin, J., 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=Efficient+cell-free+expression+with+the+endogenous+E.+coli+RNA+polymerase+and+sigma+factor+70.">Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70.</a> Journal of Biological Engineering. 4, 8 (2010).</li></ol>

J.H. is a co-founder of GenCirq Inc, which focus on cancer therapeutics. He is on the Board of Directors and has equity in GenCirq.

The protocol described here yields highly active bacterial lysate for cell-free gene expression. The key is to use cells carrying the plasmid pAD-LyseR, which expresses the lambda phage endolysin cytosolically. These cells are potentiated to lyse themselves upon permeabilization of the inner membrane, allowing the endolysin access to the cell wall, which the method achieves through a simple freeze-thaw cycle. Because the cells effectively lyse themselves, the product is referred to as autolysate. After the cells have lys...

Gene expression in cell-free lysates has several advantages over using live cells1,2,3,4. Lysates can be easily modified biochemically and used in conditions that could be detrimental to or impossible to achieve in live cells. Gene expression circuits do not have to contend or compete with host biological processes, and testing new genetic circuits is as simple as adding DNA. For these reasons, cell-free gene expression has found various applications, from biosensors5,6 to rapidly prototyping synthetic gene circuits7,8 to developing artificial cells9. Most cell-free gene expression utilizes cellular lysates that have been highly processed, generally requiring long and complex protocols, specialized equipment, and/or sensitive steps that can lead to significant variation between users and batches10,11.
This paper describes a simple, efficient method for producing cell-free lysate that requires minimal processing and expertise (Figure 1A)12. The method relies on E. coli cells that are engineered to lyse following a simple freeze-thaw cycle. The cells express an endolysin from phage lambda that degrades the cell wall. As the cells are growing, this endolysin remains in the cytoplasm, sequestered from the cell wall. However, a simple freeze-thaw cycle disrupts the cytoplasmic membrane, releasing the endolysin into the periplasm, where it degrades the cell wall, resulting in rapid cell lysis. The protocol can be completed with only a few hours of hands-on work and requires only a freezer, a centrifuge capable of 30,000 &#215; g (for optimal results; lower speeds can be used with more care not to disturb the pellet), a vortex mixer, and a simple buffer solution. Functional lysate can even be produced by freeze-drying the cells and rehydrating them in situ. However, this method produces lysates with lower activity, presumably&#160;due to the remaining cell debris.
The lysates are highly active for cell-free gene expression, and they can be enhanced in various ways depending on the end use. The rate of protein synthesis can be further increased by concentrating the lysate using standard spin concentrators. Linear DNA can be protected from degradation by adding purified GamS protein. Protein degradation, necessary for more complex circuit dynamics such as oscillation, can be achieved by co-expressing a ClpX hexamer in the autolysate-producing strain13. Finally, LacZ-based visual readouts are enabled by using an autolysate strain lacking lacZ. Overall, this method produces highly active cell-free lysate that is suitable for a wide range of applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2xYT media</td>
			<td>EMD Millipore</td>
			<td>4.85008</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>3-PGA</td>
			<td>Sigma Aldrich</td>
			<td>P8877</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filter unit, 3 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC900308</td>
			<td>optional, can be used to concentrate lysate, select concentrator capacity appropriate for the volume to be concentrated</td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A0166-5G</td>
			<td>or carbenicillin, a more stable variant</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma Aldrich</td>
			<td>A8937</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma Aldrich</td>
			<td>A9501</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>CoA</td>
			<td>Sigma Aldrich</td>
			<td>C4282</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>United States Biosciences</td>
			<td>14121</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>D-glucose (dextrose)</td>
			<td>Fisher Scientific</td>
			<td>AAA1749603</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma Aldrich</td>
			<td>D0632-1G</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>E. coli BL21-Gold (DE3) carrying pAD-LyseR</td>
			<td>Addgene</td>
			<td>99244</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BL21-Gold (DE3) &#916;lacZ carrying pAD-LyseR</td>
			<td>Addgene</td>
			<td>99245</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid</td>
			<td>Sigma Aldrich</td>
			<td>F7878</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>United States Biosciences</td>
			<td>16800</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375-25G</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Fisher Scientific</td>
			<td>DF0446075</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>magnesium glutamate</td>
			<td>Sigma Aldrich</td>
			<td>49605-250G</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>Sigma Aldrich</td>
			<td>N6522</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>potassium glutamate</td>
			<td>Sigma Aldrich</td>
			<td>G1501-100G</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>potassium hydroxide (KOH)</td>
			<td>Sigma Aldrich</td>
			<td>221473-25G</td>
			<td>for adjusting pH</td>
		</tr>
		<tr>
			<td>potassium phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma Aldrich</td>
			<td>85558</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>9310500GM</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>tRNA mix</td>
			<td>Roche</td>
			<td>10109541001</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>United States Biosciences</td>
			<td>23160</td>
			<td>or equivalent</td>
		</tr>
	</tbody>
</table>

rapid, affordable, and uncomplicated production of bacterial cell-free lysate

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

Representative results can be observed by using autolysate to express GFP from a constitutively expressing plasmid, here pBEST-OR2-OR1-Pr-UTR1-deGFP-T500, and recording a time course of GFP fluorescence in a plate reader (Figure 1B). A dilution series of plasmid DNA found strong expression even at 1 nM DNA. Compared to a commercially available lysate, the autolysate can produce a greater total yield and achieved a greater maximum production rate, calculated as the time derivative of the GFP ...

Watch this Scientific Journal Video about Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate at JoVE.com

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, ...

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Research

JoVE Journal

Biology

4.1K Views.  University of California, San Diego. This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Video: Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Construction of an Affordable and Easy-to-Build Zebrafish Facility

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

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 ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

Embedding CFPS Reactions in a Variety of Hydrogel Matrices

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Author Spotlight: A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...