我们感谢山口智子在图1绘制了示意图像。这项研究是由冈崎研究所自然科学theNational学院生物科学结合的冈崎ORION项目（NINS）的支持;由天体生物学中心项目NINS（无AB281010）;由格兰特 - 在援助来自日本学术振兴科学研究的创新领域（用于创建集成功能生物分子系统的动态排序）（JSPS）（无25102008）;由格兰特 - 在资助青年科学家（B）（以KK，没有15K17850）由日本学术振兴会; andby从JSPS格兰特 - 在援助研究活动启动（以YN，没有。15H06653）。额外的支持是由野口研究所，花王基金会艺术和科学的资助，并从栗田与环境基金会的资助拨款提供。

1.准备GVS由W / O乳化离心法<ol><li>制备1,2- dioleoyl- SN -glycero -3-磷酸胆碱的原液（DOPC，25毫摩尔）和德克萨斯红1,2- dihexadecanoyl- SN -glycero -3-磷酸的原液，三乙铵盐（德克萨斯红DHPE，0.20毫摩尔）在氯仿中并存储储备溶液在-20℃。 </li><li> 制备油溶液。 <ol><li>通过蒸发DOPC原液（51.0微升和流动的氮气下的德克萨斯红DHPE原液（19.2微升）的混合物形成5毫升玻璃小瓶的内表面上的脂质膜。 </li><li>孵育减压膜过夜，然后都加上1.0毫升的液体石蜡（0.86-0.89克/厘米3）的小瓶中。包裹在铝箔的小瓶中，并在80℃CO / N孵育它静止。 DOPC和德克萨斯红DHPE的最终浓度是1.3和3.8×10 - 3毫分别和DOPC:德克萨斯红DHPE摩尔比为100:0.3。 </li></ol></li><li> 制备内部水分散体。 <ol><li>在1.5ml带盖微型管，混合237.5微升的1.0微米的非荧光微球（2.5体积％）和12.5微升的1.0微米的荧光微球（2.5体积％）的分散体的分散体;这对应于95:非荧光的荧光微球5（V / V）的比率。 </li><li>添加64毫克蔗糖随后Tris缓冲溶液的125微升（TBS，1M）和875微升的去离子水。微球的最终体积分数为0.5体积％，和的Tris-HCl（pH为7.5）和蔗糖的最终浓度分别为0.1和0.15微米。 </li><li>涡流的微管30秒，然后超声处理它为10分钟。 </li></ol></li><li> 制备的外水溶液。 <ol><li>制备的10毫升Tris-缓冲溶液后（0.1M的Tris-HCl [pH为7.5]，0.15M GLUCOSE）中相同的操作作为内部水介质，放置1ml溶液中有盖微型管1.5毫升涡流的微管30秒，然后超声处理它为10分钟。 </li></ol></li><li> 制备含微球AW / O乳液。 <ol><li>混合1毫升油溶液（含有DOPC液体石蜡和德克萨斯红DHPE）用300μl的内水溶液的在1.5ml微管中。 </li><li>通过使用机械均化器（与该高速旋转叶片的搅拌器）乳化在微管中的两个组成部分在10,000rpm下操作在室温2分钟。 </li></ol></li><li> 沉淀的GVS。 <ol><li>轻轻在4℃在带盖微型管1.5ml的层300微升上1毫升的外水溶液的上表面上的w / o乳液中。冷却微管在4℃下10分钟。 </li></ol></li><li> 收集GVS。 <ol><li>立即冷却微管后，在30分钟18000×g的离心它。通过用图钉刺入微管的底部，并在无菌1.5毫升微型管收集一个液滴获得沉淀的GVS。 </li><li>稀释沉淀GV液滴10-100倍（体积）与外水溶液如果数量足够大，以使观测难以得到所获得的GVS。 </li></ol></li></ol> 2. GVS的显微镜观察<ol><li> 准备显微镜标本。 <ol><li>放置粘合剂孵育室用于在显微镜盖玻璃的顶部原位聚合酶链反应和杂交（室尺寸9毫米×9毫米×0.3mm厚）。 </li><li>用微量，存款25微升稀释沉淀的标本地区GVS并立即放置在孵化室的顶部的另一个玻璃盖（0.15mm左右厚）。 </li></ol></li><li> 记录微分干涉反政府GVS该效应的T型显微镜图像。 <ol><li>囊泡的记录显微镜图像用装有12V100WHAL-L卤素灯显微镜（10X，20X，和40X目标）。 </li></ol></li><li> 进行荧光显微镜观察。 <ol><li>插入U-FBNA和U-FMCHE荧光镜单元置于显微镜。适合的单位以470-495纳米和565-585纳米的激发滤光片，分别和与分别发送510-550纳米和600-690纳米的光，发射滤光片。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

<html>内部水分散介质和外侧水溶液的比重必须仔细选择。为w / o乳液，以沉淀成在进行离心处理期间外水溶液，内部水分散介质的比重必须比外水溶液大。我们试图制备使用内和外解决方案，而糖GVS，但我们得到的这些条件下没有GVS，因为内水溶液没有足够的质量，以跨越两相之间的干扰。如果有上述两种溶液，GVS该沉淀入外水溶液可以收缩或破裂之间的大的渗透压差。因此，内，外GVS渗透压必须相等。要做到这一点，我们使用蔗糖作为在内部水分散体和葡萄糖作为在外层水溶液溶质溶质;两个糖是在相同的浓度。这两种盐SS ="外部参照"&gt; 22和糖23，35，38已被用于这种目的，但通常使用的糖，因为它是毒性更小，比盐更可溶。然而，如果加入过多的糖，则GVS会与玻璃盖和崩溃的底部接触。有避免这几款策略。一种策略是降低内，外的水溶液，使得GVS温和条件下沉淀之间的比重差;具体地，沉淀的GV分散液的上清液可以用一个解决方案是相同的内水溶液通过粘接，以减少对盖玻璃作为浮力的结果囊泡破裂的可能性进行交换。另一种策略是用脂质薄膜29预涂两个盖玻片。 适当的准备和孵化乳液都重要。我们使用的液体石蜡来制备油溶液。矿物油26，31，十二烷21，33，和角鲨烷33，39也经常用作溶剂油。因为这些材料的比重，粘度和表面张力而变化，囊泡不同数量时相同的离心条件使用33甚至形成。以获得具有目标性能的囊泡，它是必不可少的，以优化比重和内外水溶液粘度以及油相的离心acceleration.For制备，孵育必须发生在高温下，在干燥环境如培养箱或脱水。在这项研究中，我们加热液体石蜡至80℃以完全溶解脂质molecules.In此外，乳剂应根据需要，应立即进行离心，因为它是不稳定的是制备后立即和仅制备的W / O型微滴容易熔合到彼此。该乳液可大量通过超声处理，涡旋，或攻丝来制备。然而，使用均化器允许大量乳液的快速制备和更容易乳化油具有高的粘度。同样重要的是，该乳液的外水溶液轻轻和迅速，然后在4℃冷却分层。缩短乳化和离心之间的时间，油外水溶液系统可以将乳液在其上层叠之前预冷，并immediately.If白浊出现比平常更快或更慢，机械整个系统然后可以离心均质必须进行彻底漂洗，以除去清洁剂。此外，乳化之前，油溶液，内水溶液和乳化剂必须为returned至室温。 适当的离心加速度也很重要。通过18000×g下离心，我们能够以高浓度包封单个内部材料制备GVS。当需要多种材料的封装，最好是减小离心加速度。例如，肌动蛋白组装系统包封个化合物，用离心低于350×g的35来实现的。在其离心是不希望的情况下，GVS可以通过调节糖浓度或通过其自身质量38，40，41的作用下沉淀该乳液得到。 本文所报导的方法有两个主要的限制。其中之一是，油分子（石蜡，在这种情况下）可以在GV膜被溶解，如已经指出的WEITZ基团21。当膜蛋白进入GV膜的插入是期望的，在蛋白质上共存的油分子的影响必须考虑。另一限制是体积分数的变化。在这项研究中，我们估计，微球在GVS的体积分数从4-30体积％范围;的体积分数不是完全相同用于GV制备的内水溶液的体积分数。虽然，我们能够在一个体积分数为显微镜观察高到足以封装在GVS微球，这种方法是不适合于制备GVS的具有均匀体积分数分布。已经报道了微球的体积分数的分布离心34期间变化。 在w / o乳液离心法通常用于含有包封材料GVS的形成。然而，很少有报道描述为PGVS微型封装材料34，42的赔偿。近日，包含封装器件的DNA或分子器件的分子机器人已建成43，44。与车厢GVS是这类应用的首选;因此，技术，如我们的，这可用于包封的磁性微球和微球与不同的表面官能可以预期是有用的44。

<html>建设性，或合成的，生物学的方法是探索生活之间的边界和非生命物质的一个迷人的途径。因为该细胞是生命所需要的最小单位，因为我们目前所知，各种研究人员试图构造简单，易于理解的化学物质的人造细胞，使这种人工细胞中发生的现象，可以研究，以阐明的最终目标生命的起源和研究活细胞1，2，3的基本功能。特别是，小泡4，它是制成两性分子球形微区和可以封装生物分子如蛋白质5,6和DNA 7，8，9，小姑娘="外部参照"&gt; 10，经常被用来作为生物膜的模型。 囊泡可以被分类为小（定义为具有直径&lt;100nm的），大（直径&lt;1微米），或巨（直径&gt; 1微米）。巨囊泡（GVS），因为它们类似于活细胞的大小，形状和结构已被广泛研究。由于GVS的大小，在GV膜的形态学变化可以很容易地进行实时的光学显微镜下观察。 制备GVS几种方法已经报道了11，包括水合方法12，13中 ，冻融法14，electroformation方法15，16和流体装置的方法17,18。然而，包封蛋白质和其它MACROM通过这些方法的装置在GVS olecules在高浓度是困难的。尤其是，它是非常具有挑战性的封装在足够量（20-30体积％）的生物材料，以模仿细胞内部19，20中的拥挤的环境。为形成GVS瞬间，韦茨和他的同事建立了一个水包油（W / O）乳液离心法21，22。这种方法有五个重要的特性。首先，因为用这种方法制备GVS具有低层数23，24，它们的膜是如此之薄，他们可以很容易地变形。由的FtsZ（细菌细胞分裂的蛋白质），微管蛋白，和其它大分子诱导GV膜变形已经研究25，26，27，28，和我们观察polyhe<html>通过微球29，30的封装诱导GV膜德龙状变形。第二，膜蛋白可以插入通过这种方法，小泡膜尽管有困难31。例如，YOMO组使用这种方法来研究膜蛋白α溶血素32的体外合成和孔形成活性。第三，有可能产生非对称GVS，其中内和外小叶的脂质组分是不同的22。例如，Whittenton 等。与内小叶来封装带负电荷的多核苷酸的阳离子脂质，并与外部小叶中性脂质产生不对称GVS降低毒性和非特异性细胞摄取33。第四，物质的GVS内的浓度和体积分数可以是相对高的S ="外部参照"&gt; 28，34。第五，多种类型的材料可封装35。例如，Nishimura 等人的封装体外转录-翻译系统进入GVS和所使用的系统的GVS 36内表达绿色荧光蛋白（GFP）。这五个特点使W / O乳化离心产生细胞模仿GVS不可缺少的方法。 在先前的工作中，通过离心分离生成的GVS通过装有用含一些最终水溶液22的长为16G的不锈钢针注射器装置收集。在经验的技术人员手中，这种收集方法很容易导致一些油的GV的污染。在这项研究中，我们使用由YOMO组23，37开发的w / o乳液离心协议其中，通过在离心管中，他们制备的底部上的孔打开沉淀GVS被收集。我们制备GVS包封1.0微米的微球，其在尺寸上与细胞内细胞器相似。使用的微球的允许我们通过计算它们的体积分数来估计其浓度。准备GVS，其中材料被密密麻麻方法的建立是创造人造细胞的重要一步。为了证实我们的各类内的材料的协议的效用，我们还证明了GFP和一个小的水溶性荧光分子（荧光素钠）可在GVS进行封装。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
			<td>Vesicular membrane molecule</td>
		</tr>
		<tr>
			<td>Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T1395MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid paraffin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>128-04375</td>
			<td>Oil, Density (20&#8451;) 0.86 -0.89 g/mL</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 7.5)</td>
			<td>Nippon Gene Co.</td>
			<td>318-90225</td>
			<td>Buffer solution</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Wako Pure Chemical Industries</td>
			<td>049-31165</td>
			<td>For outer water solution</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako Pure Chemical Industries</td>
			<td>196-00015</td>
			<td>For inner water solution</td>
		</tr>
		<tr>
			<td>Polybead carboxylate microspheres 1.0 &#181;m</td>
			<td>Polysciences</td>
			<td>08226-15</td>
			<td>Nonfluorescent, 2R = 1.0 &#181;m</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG carboxylate microspheres 1.0 &#181;m</td>
			<td>Polysciences</td>
			<td>15702-10</td>
			<td>Fluorescent, 2R = 1.0 &#181;m</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG carboxylate microspheres 0.10 &#181;m</td>
			<td>Polysciences</td>
			<td>16662-10</td>
			<td>Fluorescent, 2R = 0.10 &#181;m</td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt (uranine)</td>
			<td>Sigma Aldrich Japan</td>
			<td>F6377-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP standard (recombinant)</td>
			<td>Vector Laboratories</td>
			<td>MB-0752</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5427R</td>
			<td>Eppendorf&#160;</td>
			<td>5409000233</td>
			<td>Centrifuge rotor: FA-45-48-11</td>
		</tr>
		<tr>
			<td>Physcotron</td>
			<td>Microtec Co.</td>
			<td>NS-310EIII</td>
			<td>Handy micro-homogenizer</td>
		</tr>
		<tr>
			<td>Generator shaft</td>
			<td>Microtec Co.</td>
			<td>NS-4</td>
			<td>Homogenizer attachment</td>
		</tr>
		<tr>
			<td>Frame-Seal incubation chambers for in situ PCR and hybridization</td>
			<td>Bio-Rad Laboratories</td>
			<td>SLF0201</td>
			<td>Hybridization chamber volume: 25 &#181;L 
			Chamber size: 9 &#215; 9 &#215; 0.3 mm</td>
		</tr>
		<tr>
			<td>Frame-Seal incubation chambers for in situ PCR and hybridization</td>
			<td>Bio-Rad Laboratories</td>
			<td>SLF0601</td>
			<td>Hybridization chamber volume: 65 &#181;L&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			Chamber size: 15 &#215; 15 &#215; 0.3 mm</td>
		</tr>
		<tr>
			<td>Microman E</td>
			<td>Gilson</td>
			<td>FD10006</td>
			<td>Pipette for viscous liquid. We measured liquid paraffin by using this.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus Co.</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror unit</td>
			<td>Olympus Co.</td>
			<td>U-FBNA</td>
			<td>Excitation filter: 470-495 nm 
			Emission filter: 510-550 nm</td>
		</tr>
		<tr>
			<td>Mirror unit</td>
			<td>Olympus Co.</td>
			<td>U-FMCHE</td>
			<td>Excitation filter: 565&#8211;585 nm 
			Emission filter: 600-690 nm</td>
		</tr>
	</tbody>
</table>

preparation of giant vesicles encapsulating microspheres by centrifugation of a water-in-oil emulsion

建设性生物学和合成生物学的方法来创建人工生命涉及生物或非生物材料的自下而上装配。这种方法已经得到极大的重视科研的生活和非生命物质的之间的边界上，并已使用在过去的二十年里建造人造细胞。特别是，巨人囊泡（GVS）经常被用作人造细胞膜。在本文中，我们将介绍GVS封装高度封装的微球含有高度浓缩生物分子的细胞模型的准备。在GVS通过一个简单的水包油乳剂离心法的方法制备。具体地，均化器，使用乳化含有待包封的材料的水溶液与含有溶解的磷脂的油，将得到的乳液的另一种水溶液的表面上小心分层。然后分层系统离心ŤØ生成GVS。采用这种强大的方法来封装不等的小分子微球材料。

在w / o乳液离心法是照相并示意性示出在图1中。 图1中的概略图像表明，这种方法的成功的最重要的决定因素在于，所述内水溶液的比重必须比外水溶液的直径，从而使GVS将离心分离期间沉淀。此外，在W / O型接口的脂质单层的形成要求系统被冷却10分钟，将乳液在外水溶液分层后。因为由乳液液滴横跨的w / o接口传输的GVS形式，在内部和外部的水层的渗透压必须相同。作为对照实验，在图1示出和步骤1.3，所不同的是内水溶液我们由该方法的装置不含微球体还制备GVS无球准备。 离心后沉淀的GVS的集合示于图2。除了在微管的最底部的半透明相，我们也观察到在外层水溶液（ 图2a）的白色混浊的中间阶段。这中间阶段是丰富的微球和油的聚合，而底部阶段包含的GVS。因此，用图钉（ 图2b）刺入微管的底部后，我们只收集的首次下降（ 图2c），其中载GVS的大量。以确保不超过两个滴被收集是非常重要的;任何额外的液滴可能含有微球和脂质聚集，这将导致在GVS的较低的密度。得到的泡分散液通常载有GVS外封装材料，因为ofte的GVS离心在n个破裂。以获得仅GVS，排序应该选择一个分选方法，例如透析，凝胶过滤，或荧光激活细胞，由于包封材料的大小。如有必要，为宗旨的量，沉淀的GVS是被使用，它们可以与外水溶液稀释。在一些情况下，中间相延伸到管的底部，这表明所形成的任何囊泡是由油保持在一起。 我们得到的GVS的微分干涉显微镜和荧光显微镜图像，而不微球（ 图3A，3B）中，用微球（ 图3c -3 F）。与以前的报告23，37相一致，具有低层数GVS通过我们的协议形成。与得克萨斯红DHPE，它会发出红色fluores结合脂质萤光，使用使囊泡形成可以由薄膜的可视化可以直接证实。我们得到的160 GVS的，55包封的微球和105是空的，给予34％的包封的比率。 我们通过以下的方法来确定在GVS微球的体积分数（φ，体积％）。因为每个GV含有几十到几百微球，在光学显微镜下计数所有的微球是具有挑战性的。因此，我们用少量荧光微球，其中手动荧光显微镜下计数混合的非荧光1.0微米的微球。 （基于原始95:非荧光的5 [V / V]比荧光微球）包封的微球的总数（N）的通过用20手工计数荧光微球的数量（n）相乘来计算。的价值66;然后估计为NV 100 / V，其中v是微球的体积，V是各个GV的体积。注氮从n表示估计引起计数错误，并且这些错误必须计算φ时加以考虑。 φ值由N，将其直接计算为20 N估计，而这又导致了该φ概率 准确地反映真正的价值为&lt;50％。事实上，N波动围绕20 N一定程度上，所以我们需要把它作为20（N + i），其中i为n中的错误。我们为了使n的概率± 我可以是泊松分布的基础上，获得了超过50％的估计的I。我们估计的I，这反过来又允许我们计算20（ 正 ±i）和值66;其中包括计数误差的GVS直径的10和15微米（ 表1）。根据该过程，在图3c中所示的GV微球的体积分数估计为11±3％（体积）。所计算的体积分数的精度为10〜30％。 我们的研究结果表明，我们成功地封装在GVS 1微米的微球在高体积分数。我们使用相同的外水溶液和相同的协议（ 图4）还能够封装的其它材料为100％摩尔DOPC GVS。具体地讲，从含有0.1M的Tris-HCl（pH值7.5），0.15M蔗糖和0.5体积％的荧光微球由含有1.0微米的微球GVS描述的协议的装置（Tris-缓冲溶液制备含有0.1微米的微球GVS 图4a）。继协议中所述ABOV即，DOPC GVS含有GFP（0.1M的Tris-HCl [pH为7.5]，0.15M蔗糖，和100微克/毫升的GFP; 图4b）和含有荧光素钠GVS（0.1M的Tris-HCl [pH为7.5]，0.15M蔗糖，和30微米荧光素;还制备图4c）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ñ </td><td> ñ </td><td> φ（10微米直径） </td><td> φ（15微米直径） </td></tr><tr><td> 3 </td><td> 60±20 </td><td> 6.0±2.0 </td><td> 1.8±0.6 </td></tr><tr><td> 4 </td><td> 80±20 </td><td> 8.0±2.0 </td><td> 2.4±0.6 </td></tr><tr><td>五</td><td> 100±20 </td><td> 10±2 </td><td> 3.0±0.6 </td></tr><tr><td> 6 </td><td> 120±30 </td><td> 12±4 </td><td> 3.6±1.2 </td></tr><tr><td> 10 </td><td> 200±32 </td><td> 20±3 </td><td> 5。9±0.9 </td></tr><tr><td> 20 </td><td> 400±45 </td><td> 40±5 </td><td> 12±2 </td></tr><tr><td>三十</td><td> 600±55 </td><td> - </td><td> 18±2 </td></tr><tr><td colspan="4">如文中所述进行确定的误差; N =手工计数微球的数目; N =总数量封装的微球。 </td></tr></tbody></table> 表1: 微球上 数字和体积分数（φ， 体积％）。如文中所述进行确定的误差; N =手工计数微球的数目; N =总数量封装的微球。 <img alt="图1" src="/files/ftp_upload/55282/55282fig1.jpg" /> 图1: 流动通道<html>艺术和W / O乳化离心法示意图。 （a）由DOPC和德克萨斯红DHPE的油溶液（100:0.3摩尔比）在液体石蜡中。 （b）由蔗糖和微球体在Tris-HCl缓冲液的内水分散液。 （c）由葡萄糖在Tris-HCl缓冲液外水溶液。 （ 四 ）1毫升的油溶液和300μl的内水分散体的混合物。 （ 五 ）乳化用均化（10,000rpm下，2分钟）。 （f）在W / O乳液。 （G）于1毫升外水溶液以300μl乳液分层。 （H）沉淀GVS刚过离心。 （i）的w / o乳液离心方法的原理的示意图。黑色箭头指示的离心加速度的方向。.JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55282/55282fig2.jpg" /> 图2: 离心管穿孔。 （ 一 ）沉淀的GVS只是离心（也描述于图1小时 ）之后。红色椭圆表示沉淀的GV分散的液滴。用图钉微管（ 二 ）穿刺。通过用图钉刺入底部附近的微管，并从孔收集液滴得到含有GVS沉淀。 （c）该沉淀的GVS与外水溶液稀释分散体（由黄色箭头指示）的液滴。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig2.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:GVS 的显微图像。 （ 一 ）GV的微分干涉对比显微镜图像而没有微球。 GV的直径约为10微米。比例尺= 10微米。一个GV（b）的荧光显微镜图像而没有微球。红色荧光由德克萨斯红DHPE射出。含有微球GV（ 三 ）微分干涉对比显微镜图像。 （ 四 ）荧光显微镜的1.0微米的微球（YG羧酸微球体）一个GV内部图像。计数荧光微球（N）的数量为6，我们预计基于泊松分布错误（I = 2）。然后，我们估计内球总数（N = 20（N + I））为120±40。有人计算过，GV包含1.0微米的微球在大约11±3％（体积），其中v是，微球的体积，V是GVS的体积的体积分数（φ= NV 100 / V）。 （ 五 ）荧光显微镜一GV膜的形象。红色荧光由德克萨斯红DHPE射出。 （ 六 ）合并面板中的D和E的图像的图像。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55282/55282fig4.jpg" /> 图4:GVS 的微分干涉对比和荧光显微镜图像不同封装材料。微分干涉相衬显微镜（顶部）和荧光显微镜（下图）的图像含有（A）0.1微米的微球，（B）绿色荧光蛋白，和（c）荧光素钠GVS。比例尺= 10微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55282/55282fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion at JoVE.com

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

preparation-giant-vesicles-encapsulating-microspheres-centrifugation

Research

JoVE Journal

Biochemistry

14.4K 次观看.  Japan Women's University. Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials. 

视频: Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

氮气气蚀和差速离心允许监测外周血膜蛋白在细胞中的分布

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

高速离心条件下 u937 细胞的细胞分裂

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

巨囊泡封装微球通过一个水包油乳液的离心制备

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