APL 感谢美国国家科学基金会 （EF1935265）、美国国立卫生研究院 （R01-EB030031 和 R21-AR080363） 和陆军研究办公室 （80523-BB） 的支持

用于本研究的试剂和设备在 材料表中提供。
1. 存在小单层囊泡 （SUV） 的本体 CFE 反应
<ol><li>SUV的准备工作 注意:此步骤需要按照使用氯仿的安全说明在通风橱中执行。<ol><li>通过转移 76 μL 溶解在氯仿中的 25 mg/mL POPC 储备溶液，在玻璃小瓶中制备 5 mM 1-棕榈酰基-2-油酰基-甘油-3-磷酸胆碱 （POPC） SUV。</li>
		<li>在轻轻旋转玻璃瓶的同时，将一股温和的氩气吹入玻璃瓶中，在底部形成一层干燥脂质膜。接下来，将玻璃瓶转移到干燥器中，其盖子松动以蒸发多余的氯仿。</li>
		<li>将玻璃瓶保持在干燥器中1小时。然后，加入 0.5 mL 超纯去离子水以溶解脂质膜并涡旋约 2 分钟。</li>
		<li>通过将两个过滤器支架浸泡在去离子水中并将它们放置在每个内部膜支架上来设置微型挤出装置。然后，浸泡一个 100 nm 的聚碳酸酯过滤器，并将其放置在由挤出机外壳和固定螺母固定在一起的两个内部膜支架之间。将此设置放在挤出机支架中。</li>
		<li>用超纯去离子水冲洗两个 1 mL 气密注射器 3 次。</li>
		<li>将脂质水混合物样品装入其中一个 1 mL 气密注射器中，并使用摆臂夹将其放置在小型挤出机的一端，以将注射器固定到位。将第二个注射器插入微型挤出机的另一端，并确保其完全凹陷。 注意:当用脂质水混合物装入注射器时，请确保注射器中没有空气，然后再将其通过微型挤出机。</li>
		<li>轻轻地将脂质水混合物从原始注射器传递到空注射器中，通过微型挤出器装置。重复此步骤 11 次以形成 SUV。将 SUV 转移到 1.5 mL 微量离心管中。 注意:SUV 溶液可在 4 °C 下储存长达 2 周。</li>
	</ol></li>
	<li>CFE反应组装<ol><li>按照制造商提供的无细胞表达方案组装 CFE 反应，并稍作修改，详见下文。</li>
		<li>混合 10 μL 溶液 1（含有氨基酸、NTP、tRNA 和酶底物，以及必要的缓冲液）、1 μL 溶液 2（20% 甘油中的蛋白质）、2 μL 溶液 3（核糖体 （20 μM））、适量的编码可溶性 sfGFP-sfCherry（1-10）27（以下简称可溶性 sfGFP）、WT-GluR0-sfGFP 或 PRSP-GluR0-sfGFP（10-60 ng/1000 碱基对）， 1 μL 鼠 RNAse 抑制剂和 4 μL 5 mM SUV 溶液（用于膜蛋白）或 4 μL 水（用于可溶性蛋白）。</li>
		<li>通过加入超纯去离子水，使反应的最终体积达到 20 μL。 注意: 组装 CFE 系统时，所有组件必须保持在冰上。所有材料都对温度敏感，如果达到室温，它们就会降解。</li>
	</ol></li>
	<li>CFE反应孵育和监测<ol><li>将 CFE 溶液转移到 96 孔锥形 V 形底板中。为防止反应过程中蒸发，请使用密封膜覆盖板。</li>
		<li>将板在37°C下在酶标仪中孵育4-5小时，同时通过每2分钟测量在488nm / 528nm激发/发射波长处增益为100的GFP信号来监测CFE反应。</li>
	</ol></li>
</ol>2. 封装在GUV中的CFE反应
<ol><li>GUV外缓冲溶液的制备<ol><li>在 1.5 mL 微量离心管中，混合 1.5 μL 1 M 亚精胺、37.5 μL 100 mM ATP、25 μL 100 mM GTP、12.5 μL 100 mM CTP、12.5 μL 100 mM UTP、25 μL 1 M 磷酸肌酸、18 μL 1 M 醋酸镁、93.33 μL 3 M 谷氨酸钾， 50 μL 1 M HEPES KOH （pH 7.4）、1.15 μL 332 mM 亚叶酸、100 μL 2 M 葡萄糖和 50 μL 20 种氨基酸中每一种的 6 mM 混合物储备溶液（按照 Sun 等人描述的方案制备28）。</li>
		<li>通过加入超纯去离子水，使溶液的最终体积达到 1 mL。 注意:所有组件必须放在冰上。最后加入氨基酸混合物，以避免氨基酸耗竭28.该溶液可以以330μL等分试样进行分装，并在-20°C下储存直至使用。</li>
	</ol></li>
	<li>油中脂质混合物的制备 注意:此步骤需要按照使用氯仿的安全说明在通风橱中执行。<ol><li>在通风橱中，将 17.3 μL 25 mg/mL POPC 储备溶液和 1.08 μL 50 mg/mL 聚丁二烯-b-聚环氧乙烷 （PEO-b-PBD） 共聚物混合在 15 mL 玻璃瓶中。 注:最终的油中脂质溶液含有 0.5 mM 脂质，分别含有 95% 和 5% POPC 和 PEO-b-PBD。PEO-b-PBD 用于增强蛋白质表达过程中的膜稳定性，但保持低摩尔比以减少共聚物聚集成与脂质分子分离的胶束的倾向29,30。</li>
		<li>小心地将一股温和的氩气吹入玻璃瓶中，同时旋转小瓶以蒸发氯仿。</li>
		<li>将 1.2 mL 轻质矿物油移液到装有干燥脂质的 15 mL 玻璃瓶中。</li>
		<li>通过以最大速度涡旋 10-20 秒混合脂质和油。溶解的脂质-共聚物混合物看起来会浑浊。</li>
		<li>为确保油中所有可能的脂质聚集体完全溶解并分散在整个油中，将玻璃瓶置于50°C左右的烤箱中20分钟，然后以最大速度涡旋10-20秒。</li>
	</ol></li>
	<li>CFE反应组装和封装（图1 总结了以下步骤）。<ol><li>通过混合在步骤 2.1 中制备的 270 μL CFE 外部缓冲溶液、15 μL 5 M NaCl 和 15 μL 4.5 M KCl，在 1.5 mL 微量离心管中制备 300 μL GUV 外溶液。 注:在此步骤中，向 GUV 外溶液中加入 0.45 μL 1,4-二硫苏糖醇 （DTT）。添加 NaCl 和 KCl 的目的是调整外部溶液的渗透压以使其与内部 CFE 溶液相匹配。NaCl 和 KCl 的确切体积取决于所需的渗透压调整。可以添加 NaCl 或 KCl 或两者来调节渗透压。</li>
		<li>轻轻吸取 300 μL 油中脂质混合物，置于 GUV 外溶液顶部。 注意:添加脂质油混合物时，重要的是油不要与外层水溶液混合。添加后，油中脂质混合物和GUV外溶液之间应有一个可见的界面。</li>
		<li>将油水界面在室温下孵育 2 小时，使脂质单层形成并在界面处稳定。</li>
		<li>同时，按照第 1.2.1 节组装包含编码膜蛋白变体或可溶性 GFP 的质粒 DNA 的 CFE 反应。用 4 μL 1 M 蔗糖替换 4 μL 5 mM SUV 溶液或水。该反应将是 GUV 内溶液。 注:渗透压计用于测量内溶液和外溶液的渗透压。然后通过添加NaCl或水来相应地调节外部溶液的渗透压。CFE反应的渗透压通常约为1600 mOsm/Kg。 在反应中加入蔗糖可增加内溶液密度，从而允许囊泡在离心步骤中穿过油水界面。蔗糖的替代品是 Opti-Prep 密度梯度解决方案。</li>
		<li>将 600 μL 脂质油混合物加入含有 CFE 反应的微量离心管中，并上下剧烈移液器 ~1 分钟，以在脂质油溶液中乳化反应并在合成细胞周围形成脂质单层。 注意:最终溶液不应有任何气泡，并且应看起来不透明。</li>
		<li>在设置油水界面的 1.5 mL 微量离心管中，轻轻地将内溶液乳液移至油层顶部。 注意: 注意不要干扰或破坏界面的稳定性。</li>
		<li>在4°C下以2,000× g 离心10分钟。 注意:离心速度已针对此协议进行了优化。Adir等人31 报告了不同的离心速度。</li>
		<li>离心结束后，使用移液器小心地从微量离心管中取出多余的油和外部溶液。取出外部溶液，直到剩余体积约为 100 μL。 注意:在微量离心管的底部通常可以看到 GUV 颗粒。然而，缺乏可见的颗粒并不一定意味着没有 GUV 产量。无需使用移液器，而是可以通过抽吸去除外部溶液顶部的多余油。确保油中脂质溶液被完全去除至关重要。GUV溶液中的油污染会导致图像质量低下。</li>
		<li>轻轻地上下移液，将 GUV 沉淀重悬于剩余的 100 μL 溶液中。接下来，将 GUV 溶液转移到干净的 96 孔透明平底板中进行孵育。</li>
	</ol></li>
</ol>3. 封装的CFE反应孵育和成像
<ol><li>使用密封膜覆盖板以防止蒸发。将板在37°C孵育5-6小时。可以使用酶标仪并按照步骤 1.3.2 准备酶标仪进行孵育步骤。</li>
	<li>孵育结束后，将96孔板放在配备有EMCCD相机（或sCMOS相机），DAQ-MX控制激光器（或集成激光合路器系统）和CSU-X1转盘共聚焦（或激光扫描共聚焦）的倒置显微镜的成像台上。聚焦任何含有 GUV 的 ROI，并使用 Plan-Apochromat 60 x/1.4 NA 物镜在 488 nm 激发波长处捕获图像。</li>
	<li>将 GUV 的图像保存为.tiff格式。</li>
	<li>在图像处理软件（例如 ImageJ 或 Fiji）中打开图像。打开 亮度/对比度 设置面板。将亮度和对比度调整到适当的设置，使荧光蛋白可见。</li>
	<li>如果目标是比较不同表达蛋白质的信号强度，请首先使用位于"图像>堆栈"子菜单下的"图像到堆栈"面板堆叠包含不同蛋白质的 GUV 的单个图像。然后，使用"亮度/对比度"面板调整所有图像的亮度和对比度。</li>
</ol>

膜蛋白在巨型单层囊泡中的无细胞表达

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Y., 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=Calcium-triggered+DNA-mediated+membrane+fusion+in+synthetic+cells.">Calcium-triggered DNA-mediated membrane fusion in synthetic cells.</a> Chemical Communications. 59 (57), 8806-8809 (2023).</li><li>Moghimianavval, H., 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=Engineering+functional+membrane-membrane+interfaces+by+interspy.">Engineering functional membrane-membrane interfaces by interspy.</a> Small. 19 (13), 2202104 (2023).</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> J Vis Exp. (79), e50762 (2013).</li><li>Jacobs, M. L., Boyd, M. A., Kamat, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diblock+copolymers+enhance+folding+of+a+mechanosensitive+membrane+protein+during+cell-free+expression.">Diblock copolymers enhance folding of a mechanosensitive membrane protein during cell-free expression.</a> PNAS. 116 (10), 4031-4036 (2019).</li><li>Kostarelos, K., Tadros, T. F., Luckham, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+conjugation+of+(tri-)+block+copolymers+to+liposomes+toward+the+construction+of+sterically+stabilized+vesicle+systems.">Physical conjugation of (tri-) block copolymers to liposomes toward the construction of sterically stabilized vesicle systems.</a> Langmuir. 15 (2), 369-376 (1999).</li><li>Adir, O., 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=Preparing+protein+producing+synthetic+cells+using+cell+free+bacterial+extracts,+liposomes+and+emulsion+transfer.">Preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer.</a> J Vis Exp. (158), e60829 (2020).</li><li>van de Cauter, L., van Buren, L., Koenderink, G. H., Ganzinger, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+giant+unilamellar+vesicle+production+for+artificial+cells+-+current+challenges+and+future+directions.">Exploring giant unilamellar vesicle production for artificial cells - current challenges and future directions.</a> Small Methods. 7 (12), 2300416 (2023).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+Not+just+a+soap+opera.">Membrane proteins, lipids and detergents: Not just a soap opera.</a> Biochim Biophys Acta Biomembr. 1666 (1), 105-117 (2004).</li><li>Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent-free+systems+for+structural+studies+of+membrane+proteins.">Detergent-free systems for structural studies of membrane proteins.</a> Biochem Soc Trans. 49 (3), 1361-1374 (2021).</li><li>Bashirzadeh, Y., Wubshet, N., Litschel, T., Schwille, P., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+encapsulation+of+reconstituted+cytoskeleton+inside+giant+unilamellar+vesicles.">Rapid encapsulation of reconstituted cytoskeleton inside giant unilamellar vesicles.</a> J Vis Exp. (177), e63332 (2021).</li><li>Hwang, S. W., 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=Hybrid+vesicles+enable+mechano-responsive+hydrogel+degradation.">Hybrid vesicles enable mechano-responsive hydrogel degradation.</a> Angew Chemie Int Ed. 62 (41), e202308509 (2023).</li><li>Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+polymersomes:+a+comparative+review+towards+cell+mimicking.">Liposomes and polymersomes: a comparative review towards cell mimicking.</a> Chem Soc Rev. 47 (23), 8572-8610 (2018).</li><li>Tsumoto, K., Hayashi, Y., Tabata, J., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reverse-phase+method+revisited:+Rapid+high-yield+preparation+of+giant+unilamellar+vesicles+(GUVs)+using+emulsification+followed+by+centrifugation.">A reverse-phase method revisited: Rapid high-yield preparation of giant unilamellar vesicles (GUVs) using emulsification followed by centrifugation.</a> Colloids Surf A: Physicochem Eng Asp. 546, 74-82 (2018).</li><li>Huang, Y. L., Walker, A. S., Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photostable+silicon+rhodamine+platform+for+optical+voltage+sensing.">A photostable silicon rhodamine platform for optical voltage sensing.</a> J Am Chem Soc. 137 (33), 10767-10776 (2015).</li><li>Bailoni, E., 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=Minimal+out-of-equilibrium+metabolism+for+synthetic+cells:+A+membrane+perspective.">Minimal out-of-equilibrium metabolism for synthetic cells: A membrane perspective.</a> ACS Synth Biol. 12 (4), 922-946 (2023).</li></ol>

作者声明没有利益冲突。

实际上，任何依赖于分子或信息在细胞膜上转移的细胞过程，如细胞信号传导或细胞兴奋，都需要膜蛋白。因此，膜蛋白的重组成为实现不同应用的各种合成细胞设计的主要瓶颈。传统的去垢剂介导的生物膜中膜蛋白重建需要 GUV 生成方法，例如温和溶胀或电形成。溶胀方法通常产生小尺寸的囊泡，当复杂的溶液（在产生合成细胞时通常出现这种情况）被封装时，电形成产量显着下降<sup class="xref"...

在过去十年中，自下而上的合成生物学作为一个新兴领域越来越受到关注，在生物工程、药物递送和再生医学中具有许多潜在应用1,2。特别是，由于合成细胞的前景广阔的应用以及其促进体外生物物理研究的类细胞物理和生化特性，合成细胞作为自下而上的合成生物学的基石的发展吸引了广泛的科学界 3,4,5,6 .合成细胞通常被设计在细胞大小的巨型单层囊泡 （GUV） 中，其中重建了不同的生物过程。细胞骨架的重建 7,8、光依赖性能量再生9、细胞通讯10,11 和生物传感12 是在合成细胞中重建细胞样行为的例子。
虽然一些细胞过程依赖于可溶性蛋白质，但天然细胞的许多特性，如感应和通信，通常利用膜蛋白，包括离子通道、受体和转运蛋白。合成细胞发育的一个主要挑战是膜蛋白的重建。尽管脂质双层中膜蛋白重建的传统方法依赖于洗涤剂介导的纯化，但这些方法费力，对于对表达宿主有毒的蛋白质无效，或者通常不适合 GUV 中的膜蛋白重建13。
蛋白表达的另一种方法是无细胞表达 （CFE） 系统。CFE 系统一直是合成生物学中的强大工具，它允许使用细胞裂解物或纯化的转录翻译机器体外表达各种蛋白质14。CFE 系统也可以封装在 GUV 中，从而允许区室化的蛋白质合成反应，这些反应可以针对各种应用进行编程，例如创建光捕获合成细胞9 或机械敏感生物传感器15,16。与重组蛋白表达方法类似，膜蛋白表达在 CFE 系统中具有挑战性17。CFE 系统中的聚集、错误折叠和缺乏翻译后修饰是阻碍使用 CFE 系统成功合成膜蛋白的主要瓶颈。使用 CFE 系统进行自下而上的膜蛋白重建的困难部分是由于缺乏依赖于信号肽、信号识别颗粒、转位子和伴侣分子的复杂膜蛋白生物发生途径。然而，最近，多项研究表明，在翻译过程中微粒体或脂质体等膜结构的存在促进了膜蛋白的成功表达 18,19,20,21。此外，Eaglesfield 等人和 Steinküher 等人发现，在膜蛋白的 N 末端包含称为信号肽的特定疏水结构域可以显着改善其表达22,23。总而言之，这些研究表明，如果蛋白质翻译在GUV膜存在的情况下发生，并且使用适当的N端信号肽，则可以克服合成细胞中膜蛋白重建的挑战。
在这里，提出了一种使用重组元件（PURE）CFE反应封装蛋白质合成的方案，用于GUVs中的膜蛋白重建。选取细菌谷氨酸受体24 （GluR0）作为模型膜蛋白，研究其N端信号肽对其膜重建的影响。Eaglesfield等[22]通过构建GluR0的突变变体（称为PRSP-GluR0）来研究蛋白hododrein信号肽可提高膜蛋白重建效率的作用，并比较其表达和膜定位与野生型GluR0（以下简称WT-GluR0）携带其天然信号肽。该协议基于倒置乳液方法25 ，并进行了修改，使其对于CFE封装具有鲁棒性。在所提出的方法中，CFE反应首先使用脂质油溶液乳化，该溶液产生微米大小的液滴，这些液滴包含CFE系统并由脂质单层稳定。然后，将乳液滴分层在油水界面的顶部，该界面被另一个脂质单层饱和。然后，乳液液滴通过离心力被迫穿过油水界面。通过这个过程，液滴获得另一个单层，从而产生双层脂质囊泡。然后将含有 CFE 反应的 GUV 孵育，在此过程中膜蛋白表达并掺入 GUV 膜中。尽管该协议被指定用于GluR0的无细胞表达，但它可用于其他膜蛋白的无细胞合成或不同的合成细胞应用，如细胞骨架重建或膜融合研究26。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 nm polycarbonate filter</td>
			<td>STERLITECH</td>
			<td>1270193</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Clear Bottom Plate</td>
			<td>ThermoFisher Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy H1M Hybrid Multi-Mode Reader</td>
			<td>Agilent</td>
			<td>11-120-533</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Millipore Sigma</td>
			<td>10621714001</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1 Confocal Scanner Unit</td>
			<td>Yokogawa</td>
			<td>CSU-X1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium (Optiprep)</td>
			<td>Millipore Sigma</td>
			<td>D1556</td>
			<td>Optional to switch with sucrose in inner solution</td>
		</tr>
		<tr>
			<td>Filter supports</td>
			<td>Avanti</td>
			<td>610014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand microtubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Millipore Sigma</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>158968</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>iXon X3 camera&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid potassium salt monohydrate</td>
			<td>Millipore Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Millipore Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate&#160;</td>
			<td>Millipore Sigma</td>
			<td>M5661</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder kit (including syringe holder and extruder stand)</td>
			<td>Avanti</td>
			<td>610020</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX81 Inverted Microscope&#160;</td>
			<td>Olympus</td>
			<td>IX21</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus PlanApo N 60x Oil Microscope Objective&#160;</td>
			<td>Olympus</td>
			<td>1-U2B933&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-b-PBD</td>
			<td>Polymer Source</td>
			<td>P41745-BdEO</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-PRSP-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-sfGFP-sfCherry(1-10) plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pET28b-WT-GluR0-sfGFP plasmid DNA</td>
			<td>Homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC lipid in chloroform&#160;</td>
			<td>Avanti</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>PUREfrex 2.0</td>
			<td>Cosmo Bio USA</td>
			<td>GFK-PF201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleotide Solution Set</td>
			<td>New England BioLabs</td>
			<td>N0450</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor, Murine</td>
			<td>New England BioLabs</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>Biotechrabbit</td>
			<td>BR1401801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Millipore Sigma</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>VAPRO Vapor Pressure Osmometer Model 5600</td>
			<td>ELITechGroup</td>
			<td>VAPRO 5600</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of the bacterial glutamate receptor channel by encapsulation of a cell-free expression system

无细胞表达 （CFE） 系统是合成生物学中的强大工具，允许在合成细胞中进行细胞功能的仿生，如生物传感和能量再生。然而，重建广泛的细胞过程需要成功地将膜蛋白重建到合成细胞的膜中。虽然可溶性蛋白的表达通常在常见的CFE系统中是成功的，但合成细胞脂质双层中膜蛋白的重建已被证明具有挑战性。本文展示了一种在巨型单层囊泡（GUV）中重建模型膜蛋白（细菌谷氨酸受体（GluR0）作为模型合成细胞的方法，该方法基于在合成细胞内封装和孵育CFE反应。利用该平台，证明了用蛋白视紫红质信号肽替换GluR0的N端信号肽对GluR0成功共翻译转位到杂交GUVs膜中的作用。该方法提供了一种稳健的程序，允许在合成细胞中无细胞重建各种膜蛋白。

在包封CFE反应之前，含有天然和蛋白视紫红质信号肽的GluR0-sfGFP的两种变体（信号肽序列见 补充表1）和可溶性sfGFP在本体反应中单独表达，并通过使用酶标仪检测sfGFP信号来监测其表达（图2A).膜蛋白在不存在或存在 100 nm SUV 的情况下表达。此外，使用将sfGFP信号与其浓度相关联的校准曲线（补充图1），估计合成蛋白质的浓度（补?...

Watch this Scientific Journal Video about Membrane Protein Reconstitution in Synthetic Cells at JoVE.com

作者聚焦:合成细胞中的膜蛋白重构

该协议描述了用于在巨大的单层囊泡（GUV）内封装无细胞表达（CFE）系统的倒置乳液方法，用于研究模型膜蛋白的合成和掺入脂质双层。

通过封装无细胞表达系统重建细菌谷氨酸受体通道

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

我们的方案解决了使用倒滴乳液方法制成的合成细胞脂质双层内膜蛋白的重建。使用倒滴乳液方法的优点是它能够产生合成细胞，为膜蛋白的无细胞表达提供稳定的环境。我们的方案为该领域的研究人员提供了一种强大的方法，可以使用无细胞表达系统在合成细胞内合成膜蛋白。我的实验室正在研究通过一组明确定义的离子通道重建动作电位来重建合成细胞中的各种离子通道。

reconstitution-bacterial-glutamate-receptor-channel-encapsulation

Research

JoVE Journal

Bioengineering

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

1.0K 次观看.  University of Michigan, Ann Arbor. 我们的方案解决了使用倒滴乳液方法制成的合成细胞脂质双层内膜蛋白的重建。使用倒滴乳液方法的优点是它能够产生合成细胞，为膜蛋白的无细胞表达提供稳定的环境。我们的方案为该领域的研究人员提供了一种强大的方法，可以使用无细胞表达系统在合成细胞内合成膜蛋白。我的实验室正在研究通过一组明确定义的离子通道重建动作电位来重建合成细胞中的各种离?...

视频: 作者聚焦:合成细胞中的膜蛋白重构

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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.

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

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.

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

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

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

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.

快速生长的纳氏弧菌的无细胞蛋白表达

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

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...