Name: rapid, affordable, and uncomplicated production of bacterial cell-free lysate
Uploaded: 2021-10-29T14:00:00
Description: Watch this Scientific Journal Video about Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate at JoVE.com

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-...

<ol>
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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>

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, ...

Using this protocol, high-quality bacterial cell lysates can be produced using only broadly available common laboratory equipment, making cell-free gene expression easily accessible to most researchers. Combining program cellular autolysis with a freeze-thaw or freeze-dry cycle allows creating a practical, labor and cost effective approach for rapid production of bacterial lysates for cell-free gene expression. Begin by using an inoculation loop to streak the cells of an autolysis E.coli strain onto LB agar plates containing 50 micrograms per milliliter ampicillin, then incubate the plates at 37 degrees Celsius overnight.The following day, using a pipette tip, pick a single colony from the agar plate and add it into a culture tube containing LB medium with ampicillin. Grow this starter culture at 37 degrees Celsius overnight. The next day, add 400 microliters of the starter culture into a one liter Erlenmeyer flask containing 400 milliliters of 2xYTPG medium supplemented with 50 micrograms per milliliter ampicillin.Incubate the culture at 37 degrees Celsius with shaking at 300 RPM. Using a spectrophotometer and an optical cuvette with a one centimeter path length, periodically measure the culture's optical density at 600 nanometers. When the optical density exceeds one, begin diluting the culture five-fold before measurements to ensure that the measurements remain within the linear range of a typical laboratory spectrophotometer.Once the optical density of the five-fold diluted culture reaches 0.3, remove the flask from the incubator and proceed to prepare the lysate. To prepare the lysate, harvest the cells by centrifugation at 1, 800 x g for 15 minutes at room temperature. Pour off the supernatant and remove the remaining liquid using a pipette.Add 45 milliliters of cold S30A buffer to the pellet and resuspend the pellet by vortexing. Then, weigh an empty 50 milliliter centrifuge tube before transferring the cells into the tube. Centrifuge the cells again and discard the supernatant.Aspirate any remaining supernatant using a pipette. Weigh the tube again and subtract the weight of the empty tube from the weight displayed to obtain the weight of the pellet. For every one milligram of cell pellet, add two microliters of cold S30A buffer supplemented with two millimolar dithiothreitol.Then, resuspend the cells by vigorous vortexing. Next, freeze the cells by placing them in a negative 20 or negative 80 degrees Celsius freezer until the pellet is thoroughly frozen, then thaw the cells in a room temperature water bath and vortex vigorously for two to three minutes. Incubate the cells at 37 degrees Celsius for 45 minutes with shaking at 300 RPM, then clear the sample of heavy cellular debris by centrifuging in transparent centrifuge tubes at 30, 000 x g for 45 minutes at four degrees Celsius.Carefully transfer the supernatant to a new tube with a pipette, taking care not to disturb the pellet. If the transferred supernatant is contaminated with material from the pellet, repeat the centrifugation. Transfer the supernatant to 1.5 milliliter centrifuge tubes.And centrifuge once more at 21, 000 x g or the maximum speed of a tabletop centrifuge for five minutes. Aliquot the cleared autolysate into desired volumes and store at minus 80 degrees Celsius until use. For cell-free gene expression using the autolysate, mix eight microliters of autolysate and 8.9 microliters of pre-mix on ice.Add DNA to a final concentration of eight nanomoles, any other necessary reagents and water to obtain a final volume of 20 microliters. Add the reaction to a 384-well microplate and use a plate reader to measure the fluorescence time course and endpoints. GFP expression using the autolysate with a dilution series of plasma DNA results in strong expression even with one nanomolar DNA.Upon comparing the GFP expression between a commercially available lysate and the autolysate, the autolysate produced comparable levels of GFP. Variability between batches of lysate produced in two different labs by different researchers was within about two-fold. The three most critical variables are cell pellet to buffer ratio, transferring debris-free supernatant and using optimal PEG and magnesium concentration in the final reaction.Once the extract has been prepared, a large arsenal of existing protocols developed for E.coli cell-free expression systems can be used, such as ClpXP-mediated degradation, or AHL-mediated quorum sensing.

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