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

作者感谢Zachary Sun和Richard Murray（加州理工学院）提供了质粒P_araBAD-gamS，并感谢Kaeko Kamei（京都工业大学）提供了高速冷却离心机。这项工作得到了美国国立卫生研究院和ARO MURI计划的资助，并得到了日本文部科学省优秀青年研究人员领先倡议的部分支持。

1. 准备培养基和缓冲液。
<ol><li>准备 2xYTPG 培养基。<ol>
<li>将62克2xYT粉末，5.99克磷酸钾单碱，13.93克磷酸氢钾和去离子水混合至2升。</li>
		<li>在液体循环上高压灭菌，暴露时间为30分钟14。</li>
		<li>从1.1.2到400mL的2xYTP培养基，加入7.2g D-葡萄糖（葡萄糖）并混合直至溶解。</li>
		<li>通过0.2μm过滤器进行过滤灭菌。</li>
	</ol>
</li>
	<li>准备 S30A 缓冲液。<ol>
<li>混合Tris-HCl（pH 7.7，50 mM?...

<ol>
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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>

这里描述的方案产生用于无细胞基因表达的高活性细菌裂解物。关键是使用携带质粒pAD-LyseR的细胞，其以胞质方式表达lambda噬菌体内溶蛋白。这些细胞被增强以在内膜的通透性下裂解自身，允许内溶蛋白进入细胞壁，该方法通过简单的冻融循环实现。由于细胞有效地裂解自身，因此该产物被称为自溶物。细胞裂解后，仅剩的步骤是孵育和离心以清除细胞碎片的自溶物。
与其?...

与使用活细胞1，2，3，4相比，无细胞裂解物中的基因表达有几个优点。裂解物可以很容易地进行生物化学修饰，并用于可能对活细胞有害或不可能实现的条件下。基因表达回路不必与宿主生物过程竞争或竞争，测试新的遗传回路就像添加DNA一样简单。由于这些原因，无细胞基因表达已经找到了各种应用，从生物传感器5，6到快速原型合成基因电路7，8到发育中的人造细胞9。大多数无细胞基因表达利用经过高度处理的细胞裂解物，通常需要漫长而复杂的实验方案，专用设备和/或敏感步骤，这可能导致使用者和批次之间的显着差异10，11。
本文描述了一种简单、有效的无细胞裂解物生产方法，该方法需要最少的处理和专业知识（图1A）12。该方法依赖于大肠杆菌细胞，这些细胞被设计为在简单的冻融循环后裂解。细胞从噬菌体λ中表达一种内溶解素，该内溶蛋白会降解细胞壁。随着细胞的生长，这种内溶蛋白保留在细胞质中，与细胞壁隔离。然而，一个简单的冻融循环会破坏细胞质膜，将内溶蛋白释放到周质中，在那里它会降解细胞壁，从而导致快速细胞裂解。该协议只需几个小时的动手工作即可完成，只需要一个冰箱，一个能够30，000×克的离心机（以获得最佳结果;可以使用较低的速度，更小心地不要干扰沉淀），一个涡旋混合器和一个简单的缓冲溶液。功能性裂解物甚至可以通过冷冻干燥细胞并原位再水化来产生 。然而，这种方法产生活性较低的裂解物，可能是由于剩余的细胞碎片。
裂解物对无细胞基因表达具有高度活性，并且可以根据最终用途以各种方式增强。通过使用标准离心浓缩器浓缩裂解物，可以进一步提高蛋白质合成速率。通过添加纯化的GamS蛋白，可以保护线性DNA免于降解。蛋白质降解是振荡等更复杂的电路动力学所必需的，可以通过在产生自洽物的菌株13中共表达ClpX六聚体来实现。最后，通过使用缺乏lacZ的自溶物菌株来启用基于 LacZ的视觉读数。总体而言，该方法可产生高活性的无细胞裂解物，适用于广泛的应用。

<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

无细胞基因表达提供了生物学的力量，而没有生物体的并发症。尽管存在许多这样的基因表达系统，但大多数购买和/或需要特殊设备和精细磨练的专业知识才能有效生产。该协议描述了一种生产细菌无细胞裂解物的方法，该裂解物仅使用标准实验室设备并且需要最少的处理， 支持高水平的基因表达。该方法使用大肠杆菌菌株产生不影响生长的内溶素，但在简单的冻融循环后有效地裂解收获的细胞沉淀。唯一需要的进一步处理是短暂的孵育，然后离心以清除细胞碎片的自溶物。动态基因回路可以通过收获前细胞中ClpX蛋白酶的异源表达来实现。缺乏lacZ基因的大肠杆菌菌株可用于使用比色法或荧光读数的高灵敏度，无细胞生物传感应用。整个方案只需要8-9个小时，从接种到完成只需要1-2个小时的动手劳动。通过降低获得无细胞裂解物的成本和时间，该方法应提高各种应用中无细胞基因表达的可负担性。

通过使用自溶物从本构表达质粒中表达GFP，可以观察到具有代表性的结果，这里pBEST-OR2-OR1-Pr-UTR1-deGFP-T500，并在读板器中记录GFP荧光的时间过程（图1B）。质粒DNA的稀释系列即使在1 nM DNA下也有很强的表达。与市售裂解物相比，自溶物可以产生更大的总产量并实现更大的最大生产率，计算为GFP时间过程的时间导数（图1C，D）。有关使用此方?...

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

该协议描述了一种快速而简单的方法，使用工程化的 大肠杆菌 菌株生产细菌裂解物以实现无细胞基因表达，并且只需要标准的实验室设备。

快速、经济、简单地生产无细菌细胞裂解物

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

使用该协议，仅使用广泛使用的通用实验室设备即可生产高质量的细菌细胞裂解物，使大多数研究人员可以轻松获得无细胞基因表达。将程序细胞自溶与冻融或冻干循环相结合，可以创建一种实用，实用且具有成本效益的方法，用于快速生产用于无细胞基因表达的细菌裂解物。首先使用接种环将自溶大肠杆菌菌株的细胞划到含有每毫升50微克氨苄西林的LB琼脂平板上，然后将板在37摄氏度下孵育过夜。第二天，使用移液器吸头，从琼脂平板中挑选单个菌落，并将其加入含有氨苄西林的LB培养基的培养管中。在37摄氏度下过夜种植这种发酵剂。第二天，将400微升的起始培养物加入一升锥形瓶中，该烧瓶含有400毫升2xYTPG培养基，每毫升补充50微克氨苄西林。在37摄氏度下孵育培养物，以300 RPM振荡。使用分光光度计和具有1厘米路径长度的光学比色皿，定期测量培养物在600纳米处的光密度。当光密度超过1时，在测量前开始将培养物稀释五倍，以确保测量保持在典型实验室分光光度计的线性范围内。一旦五倍稀释培养物的光密度达到0.3，从培养箱中取出烧瓶并继续制备裂解物。为了制备裂解物，通过在室温下以1， 800×g离心15分钟来收获细胞。倒出上清液，用移液管除去剩余的液体。向沉淀中加入45毫升冷S30A缓冲液，并通过涡旋重悬沉淀。然后，称量空的50毫升离心管，然后将细胞转移到管中。再次离心细胞并丢弃上清液。使用移液器吸出任何剩余的上清液。再次称量管子，并从显示的重量中减去空管的重量，以获得沉淀的重量。对于每一毫克细胞沉淀，加入两微升冷的S30A缓冲液，并补充两毫摩尔二硫甲状腺素。然后，通过剧烈涡旋重悬细胞。接下来，通过将细胞置于负20或负80摄氏度的冰箱中冷冻细胞，直到沉淀完全冷冻，然后在室温水浴中解冻细胞并剧烈涡旋两到三分钟。将细胞在37摄氏度下孵育45分钟，以300RPM振荡，然后通过在透明离心管中以30，000×g在4摄氏度下离心45分钟来清除样品中的重细胞碎片。用移液管小心地将上清液转移到新管中，注意不要干扰沉淀。如果转移的上清液被沉淀中的物质污染，则重复离心。将上清液转移到1.5毫升离心管中。并以21， 000 x g或台式离心机的最大速度再次离心五分钟。将清除的自解物等分到所需的体积中，并在零下80摄氏度下储存直至使用。对于使用自溶物的无细胞基因表达，在冰上混合8微升自体溶物和8.9微升预混液。将DNA加入8纳摩尔的终浓度，任何其他必要的试剂和水，以获得20微升的最终体积。将反应加入384孔微孔板中，并使用读板仪测量荧光时间过程和终点。使用具有稀释系列血浆DNA的自溶物进行GFP表达，即使使用一个纳摩尔DNA也能产生强烈的表达。在比较市售裂解物和自溶物之间的GFP表达时，自溶物产生了相当水平的GFP。由不同研究人员在两个不同的实验室生产的裂解物批次之间的差异大约在两倍之内。三个最关键的变量是细胞沉淀与缓冲液的比例，转移无碎屑的上清液以及在最终反应中使用最佳的PEG和镁浓度。一旦制备出提取物，就可以使用为无大肠杆菌无细胞表达系统开发的大量现有方案，例如ClpXP介导的降解或AHL介导的群体感应。

Research

JoVE Journal

Biology

Maintaining Wolbachia in Cell-free Medium

维持细胞培养基的Wolbachia

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

科研

生物学

Assaying Proteasomal Degradation in a Cell-free System in Plants

测定蛋白酶体降解的植物无细胞系统

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

一个经济实惠，易于构建斑马鱼设施建设

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

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

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

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

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

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

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

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

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

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)

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

硬骨模型日本青鳉鱼（Oryzias latipes）中的精子收集和计算机辅助精子分析

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

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

大肠杆菌抗生素持久性的群体和单细胞分析

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

A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels (Video) | JoVE

在宏观水凝胶中包埋无细胞蛋白质合成反应的方法

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