We thank Dr. Mitch Doktycz, Dr. Jennifer Morrel-Falvey, Dr. Amber Bible, and Dr. Brandon Razooky for helpful advice and conversations, and acknowledge Dr. Sukanya Iyer for constructing the Pet3a-EGFP plasmid used in the gene expression tests. We acknowledge support from the Center for Nanophase Materials Sciences, which is sponsored by the Scientific User Facilities Division, Office of Science, U.S. Department of Energy. SEN and PMC acknowledge support from Bredesen Center Fellowships at the University of Tennessee, Knoxville. This research was performed at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT-Battelle, LLC, for the U.S. Department of Energy.

对设备大师1.光刻<ol><li>脱水的热板上干净的硅晶片，在〜250℃下进行至少1小时。 注：这是准备一个高手，如果用户错误时使用一个以上晶圆很好的做法。 </li><li>准备光刻胶等分。准备两个SU-8 2015抗蚀剂和稀释的SU-8 2015抗蚀剂在2的等分试样：1的比例用SU-8稀释剂作为稀释剂。 注释：大约需要为旋涂一个晶片1毫升光致抗蚀剂。 </li><li>准备三道光罩图案用于生产这些大师。对于膜万事达，准备两个掩模：1图案形成膜通道和其他图案化的反应室。对于控制阀的主，备只有一个光罩图案。 注意：有关的光刻技术的更多细节，包括掩模图案化，参见伊藤和冈崎，2000。36见Fowlkes和科利尔，2013为器件设计的更详细的描述37。 </li><li>制备膜主<ol><li>旋涂2：1的SU-8 2015抗蚀剂稀释在晶片上以1000rpm进行45秒。 </li><li>软烘烤晶片在95℃下2分钟。使用接触对准器，露出晶片膜通道模式下10秒，并在95℃下进行曝光后烘烤2分钟。 </li><li>开发晶片中的SU-8显影剂1分钟，或直至光刻胶残留物去除。晶圆清洗用异丙醇，从上移动到底部。干燥晶片用氮气，再移动从上到下。烘烤的晶片在180℃下进行4分钟。 </li><li>旋涂再图案化的晶片用2:1 SU-8稀释以2000rpm，持续45秒。 </li><li>软烘烤在95℃下图案化的晶片2分钟。用接触对准，对准图案化晶片的反应室中的图案，并暴露持续10秒。为2分钟，在95℃下进行曝光后烘烤。 </li><li>如在步骤1.4.3描述开发晶片。显影和干燥晶片，烘烤晶片在180℃下进行4分钟后。 注：该晶片可以在在前面的步骤中使用的相同的显影剂来开发。 </li></ol></li><li>准备控制阀主<ol><li>旋涂未稀释的SU-8光致抗蚀剂上的清洁晶片以2000rpm，持续45秒。 </li><li>软烘烤晶片在95℃下进行6分钟。使用接触对准器，暴露出晶片与控制阀图案10秒。执行曝光后烘烤在95℃下进行6分钟。 </li><li>开发晶片中的SU-8开发为2分钟，或直至残留物除去。冲洗用异丙醇，从顶部移到底部。干燥晶片用氮气和烘烤，在180℃进行4分钟。 </li></ol></li></ol> 2. PDMS设备制造<ol><li> Silanize所有与主人通过〜气相沉积0.2毫升三甲基氯硅烷。 <ol><li>在室温迅速围住主在密闭容器中有几滴硅烷化剂。 注：其他硅烷化协议可以接受38如果执行properly，则PDMS将是容易清除。 </li></ol></li><li>混合市售聚（二甲基硅氧烷）（PDMS）基和固化剂以不同比例的设备的两者的膜和控制阀的层，如已被证明在相同的多层阀设计39。使用20：1和5：碱比例1：固化剂为膜和控制阀模具，分别。 <ol><li>对于膜模具，混合10克基础与0.5克固化剂。 注：此卷将被旋涂在膜上的主人。 </li><li>用于控制阀的模具，混合，在5的基料和固化剂：1的比例。必要模制控制阀将取决于用来保持控制阀的主容器上的PDMS的量;填充容器，使得所述主涂覆有约1cm的PDMS。 </li></ol></li><li>调匀既PDMS制剂，和去气它们在真空室中，直到没有气泡可见。将控制阀在主耐热容器中，诸如玻璃盘。仔细倒入5：1的比例PDMS过主人，脱气容器中的第二次。 </li><li>而控制阀的PDMS容器被脱气，旋涂在20：在膜主1的比例的PDMS由PDMS混合物小心地倾倒在膜上主尽量减少气泡的形成，然后旋涂在主以1,000rpm为45秒。 </li><li>局部固化两者的主人在烘箱在80℃下进行6分对膜主至15分对控制阀的主人。 备注：当的部分固化的PDMS应该保持其形式，但材料会稍微粘性。如果PDMS尚未固化，再次烘烤以在一个时间几分钟递增直至按下时该材料保持其形式。 </li><li>从削减控制阀主矩形PDMS模具，剥离模具走平缓。使用0.75毫米打孔打穿成型部件进气孔。 注：该孔可以通过插入23号blun待清洁吨尖端的针，和在模具外部可以用玻璃纸胶带进行清洁，如果需要的话。 </li><li>使用光学显微镜，以定位在所述膜主反应室，对准与反应室的膜的特性的控制阀的模具组件，并直接放置在控制阀组件上的主膜的顶部。定向控制阀入口到装置的左下角，并确保该反应室及该膜的主通道是矩形的控制阀内可见。 </li><li>烤对准模具零件在80℃2小时。 注意：膜和控制阀的模具现在将密封在一起，并操纵作为一个模具。 </li><li>切割层状的PDMS模具离膜的主人，剥离模具远离主轻轻以免穿孔膜。 </li><li>冲床入口和出口孔，使用0.75毫米的孔打孔细胞提取物输入。通过两层打孔，并如步骤2.6中所述清理它们以相同的方式。 </li><li>使用感应耦合等离子体清洁器，等离子体处理二者的模具（膜面朝上），并在10.5瓦20秒的第0号玻璃盖玻片。立即删除来自等离子体清洁器的盖玻片和模具和层组件，膜侧朝向玻璃，试图最小化玻璃和模具之间的空气袋。不直接压在膜的输入通道，或膜可退火到玻璃上，使其难以填充反应物的通道。 <ol><li>搬运组装设备时，避免打破玻璃层要特别小心。使用薄的玻璃盖玻片作为装置必须通过采用高倍率油浸目标的玻璃盖玻片被成像 - 如果玻璃太厚，该装置的特征可以是不可见的。 </li></ol></li><li>最后，固化的完成装置，在80℃进行2小时。 </li></ol> 3.实验FO设置ř无细胞蛋白质合成反应<ol><li>通过煮沸它在去离子水中1小时水合的装置。 注：设备应该有一个阴天的外观，当完全水合。装置还可以以水合物它留下的O / N的无菌水在RT。 </li><li>使用倒置显微镜与孵育室，设置环境温度至30℃。 注意：此温度被选择以优化的GFP表达T7启动子，所以最佳温度为其它反应可能会发生变化40。 </li><li>安装设备显微镜阶段持有人用透明胶带和包装，以维持当地的水合装置用湿纸巾的边缘。 </li><li>使用两个高精度闭环电压压力传感器来调制用于控制阀的致动和试剂输入氮气压力。 注：此协议只被测试与低纯度氮，但是也可使用其它惰性气体。 <ol><li>通过连接第一换能器24号PTFE管，以用隔膜盖4毫升玻璃小瓶中保持一个水容器。所述贮存器连接到使用的第二管通过一个23号钝针头尖端终止了控制阀的入口。 注：两个管渗透油藏隔了两个尖锐的23号针。 </li><li> 24号PTFE管连接到一个男性与男性露儿乐接口连接第二传感器。附加给鲁尔乐23号针头通过与另一23号钝头针头，它被单独组装每个设备管道相连。这个针连接到膜反应通道;用它来从反应信道和输入试剂冲洗水。 </li></ol></li><li>使用无细胞蛋白质表达系统，组装的部件，在冰上的CFPS反应，根据制造商的说明。最小化花费保持CFPS试剂在冰上的时间和组装后，立即将反应到器件中。 注释：本设备已经用市售大肠杆菌中使用大肠杆菌提取蛋白的表达工具包和一个质粒组成型表达GFP。总反应体积缩放到25微升 - 这可能是可以使用甚至更低的量为反应物，如果需要的话。作为CFPS试剂倾向于敏感冻融循环，它可能有助于在之前的实验中，合适的体积，使该试剂的等分试样。其它试剂可以加入到反应混合物中，但反应必须被施加到设备之前被完全组装。 <ol><li>组装反应最后加入的DNA的输入。 注意：一旦组装在Eppendorf管中，CFPS反应将开始如果不保持在冰上。由于所需的时间的试剂适用于在设备和开始实验可以变化，它是有帮助的启动计时器一旦反应被组装并混合 - 这将维持一致的实验之间的时间刻度以及援助在排除故障。 </li></ol></li><li>使用在步骤3.4.2中所述油管和针连接器，使用1ml注射器收回组装反应进入管。插入钝头针头进入反应室的入口。从注射器卸下针连接器，并将其连接到公对公连接器用于反应室传感器。 </li><li>施加压力（&lt;10磅）到CFPS反应物来填充通道。取出针时，反应被充满。 </li><li>来自其它换能器到控制阀入口插入钝管。禁止增压控制阀呢。 </li><li>放置在安装设备上的阶段。采用明场成像，定位反应室用100X油浸物镜。 </li><li>致动通过加压控制阀换能器20 psi的控制阀;在该膜的可见光的变化将是显而易见的，当控制阀被致动。专注的反应室的底部。 </li><li>开始图像采集;生长在荧光瓦特病是在内部和反应室的外部周围可见的，尽管它可能并不明显，在反应的早期阶段。捕捉图像每隔1-3分钟，直到反应达到稳定状态荧光。如果自动聚焦阶段是不可用，简要地重新聚焦之前，被拍摄的图像的每个图像。 注意：虽然将发生某些光漂白，可占只要光漂白的速率是已知的，由于光漂白上相对荧光的影响。这种光漂白速率可以通过暴露的荧光标准，如GFP的已知浓度或荧光团的混合物，以恒定的光漂白过一段时间来估计。 </li><li>录从反应组件经过对获取的第一图像的时间。 注：这通常需要4-5分钟。 </li></ol> 4.图像分析和数据处理<ol><li>使用图像分析软件如ImageJ的，选择反应室作为ROI内部。获得投资回报的所有图像的平均荧光强度值。 注：这是原始的荧光强度跟踪。 <ol><li>执行中的ImageJ此任务使用的时间序列分析仪和投资回报率管理器插件 - 配以时间系列分析仪来选择的围绕每个反应室的内部感兴趣的区域。设置“AutoROIProperties”发送对应于每个反应室的内部的区域，检查“添加点击”，并选择每个室中。 注意：此步骤也可使用椭圆工具周围绘制荧光腔的ROI进行。此ROI大小通常对应于一个30×30象素椭圆用于与100X物镜观察到的10微米直径室。 </li><li>突出显示所有ROI的ROI经理。使用“多测量”功能通过整个图像堆栈，以确定每个ROI的荧光强度平均值。 注：命名站一个插件ckReg可以用于对准图像堆栈如果必要。 </li></ol></li><li>获取原始荧光强度的痕迹在一个实验中所有的腔室之后，通过取一个跨实验均在所有的痕迹，并减去从个别原料痕迹的平均值确定该反应的确定性分量。利用数据分析软件，如IGOR或MS Excel进行这种分析。 注意：这提供了噪声跟踪对于每个反应室。 </li><li>分析来自使用用于分析从细胞25衍生的基因表达的噪声的相同方法，这些反应室的基因表达的噪声。 </li></ol>

The authors have nothing to disclose.

在细胞中的基因表达本身嘈杂由于小细胞体积和重要的反应物的低拷贝数。噪声生物学往往集中于源，处理，并在人群中，浓度，位置或控制基因电路和网络44分子的状态波动的生物后果。这项工作的绝大多数已在蜂窝系统中，其具有观察细胞内的基因网络的天然环境中的基因电路的噪声的优点已经执行。但是，无细胞系统允许个人基因电路的固有波动没有混杂外在影响18不能避免在蜂窝系统的表征。噪声分析可以提供重要的物理见解，他们怎么电路基因的结构和如何运作，并已应用在蜂窝系统表征负25和积极<suP&gt; 27自动调节，外在和表达的噪音18，和转录内在贡献爆棚45,46。在这里，我们描述了在微流体装置，使同时控制反应器的大小和反应起始时间无细胞表达系统的研究中，为了更好地理解，分娩和拥挤47,48对内在蛋白表达的噪声，而不对所述角色并发症与活细胞相关联。 键使设计的特征是飞升容积（微米级），用于限制无细胞蛋白质表达系统的反应物，以在PDMS弹性体“控制阀”膜捕获该反应室的阵列的集成在对于反应开始一个明确定义，“时间零点”的反应物（ 图1）。这种控制允许参与蛋白质合成的反应的动力学可以FOLL欠实时高精度。正因为如此，它管理的无细胞的反应物是很重要的，使得跨实验可变性最小化，尽可能。这个控制使我们能够评估的方式，类似于先前技术用于评估在活细胞中的基因表达的无细胞的遗传电路的噪声的结构。 如在CFPS系统中使用的反应物可以是敏感的冻融循环，以保持反应物冷，并尽量减少反应物花费解冻冰的时间是很重要的。它是为了识别随时间的改变的表达水平，以定期测试散装CFPS系统的表达良好做法 - 这可能在在Eppendorf管中的10-15微升反应来完成，或者在像酶标仪设备，它执行多次读取随着时间的推移捕捉反应动力学。注意到反应物的年龄和解冻时间为每个实验可以帮助排除故障时，低表达升evels。此外，装配CFPS试剂时，要注意的是，反应开始，一旦它被完全组装并从冰除去是很重要的。为了保持一致的“零时间”，它是有帮助的，记录的时间之后的最后加入的DNA的输入的后CFPS反应开始，并尽可能快地应用该反应向保温装置。这个过程大约需要4-5分钟，荧光不应该还可以在反应室可见。这种控制确保了可利用的时间可视化的反应曲线的生长部分最大化。 在设备上运行CFPS反应之前，最好是运行质量控制测试，以验证不存在来自所述腔室的泄漏。甲FRAP测试可以通过将荧光团给设备和曝光单个井，直到井完全漂白来执行（如在图2D）。如果是室密封良好，没有恢复应该是可见的井内​​ - 应该有隔室的壁和内部和外部空间之间形成了鲜明的对比。如果荧光恢复是明显或反应室的壁没有明确定义，在控制阀中的压力应增加或设备应该检查泄漏或脱层从载玻片上。 该协议已经过测试CFPS试剂从商业E.大肠杆菌无细胞蛋白质表达试剂盒（缩放到25微升），尽管其它健壮CFPS系统都可以使用。因此能够施加反应设备，这可能是有益的，当试剂的成本是在实验中的一个限制因素时要使用的卷远低于25微升。一旦反应物添加到设备和反应室是密封的，这是不可能的反应物添加到该溶液中，而不去致动控制阀 - 因而该装置不suitab乐为反应需要在反应过程中加入试剂。脱水和器件的干燥后的这段时间内的效果不被评为 - 该设备还没有用于观察其可以比3小时运行时间CFPS反应优化。如果较长的反应时间是所希望的，这些影响可能通过密封装置以防止蒸发，改变培养温度来减轻，或通过使用湿室中。修改到装置的设计，例如纳米多孔结构的腔室壁49,50或包含多孔质膜层的，代表一些方法，它们可以允许试剂交换，从而延长反应时间尺度。 均匀量的微加工的反应车厢是保持整个实验，非常适合进行调查一致的尺寸为“不良反应”与车厢壁有价值。与使用方法不正微细加工技术，这些反应必须在小数字进行评估，并且在实验过程中不提供立体的灵活性。然而，可控设计用于这些反应室是非常适合的时间推移显微镜，并且可以是照明补充约束的高通量方法。

无细胞系统提供用于观察生物反应不受复杂因素如健身，除法和突变是不可避免的在活细胞中的研究的简化和灵活的平台。这样的方法已被用来研究细胞系统，包括膜蛋白1的表征，蛋白质相互作用2的探测，并翻译3-7的基本方面的探索。最近无细胞系统已经开始站稳脚跟的可行平台合成生物学8-10。这种方法的吸引力在于，他们摆脱了资源共享和“外在噪音”影响的反应动力学在活细胞中合成生物学。然而问题仍然对如何在其中无细胞反应被嵌入的物理环境影响反应的进展和结果。无细胞反应的环境 - 尤其是密闭environm经济需求测试的接近电池相关卷 - 仍然很差特点。无细胞蛋白质合成（CFPS）是作为是“无尺度”，通过一系列微升至升规模的反应体积11的参展相当于动力学的传统思想。尽管如此，围反应蜂窝刻度卷已被证明显著影响蛋白表达率12。 无细胞反应的随机性质 - 尤其是这些系统的方法甚至还要低于飞升卷 - 可能是特别重要的。噪声中的基因表达是通过限制为小细胞体积和成分的高密度极大地影响了一个属性迫使许多重要的分子以非常低的人口水平-例如，一个1 FL体积内大肠杆菌范围多达4300不同多肽下几百个不同的启动子1的诱导控制3。这种固有的噪声已被暗示为在许多生物过程，包括趋化性14的中央的驱动力，主动复制及延迟15，裂解和溶原16,17之间的λ噬菌体的决定，和能力之间的subtillus芽孢决定之间的艾滋病毒的决定和产孢17。无细胞合成生物学则提供了一个机会，探索细胞基因电路和网络的随机特性，并操纵这些行为，以达到特定的技术目标。而蜂窝系统的噪音行为已被充分研究的18-27，出现了小的探索无细胞系统8的基本噪声特性，特别是在细胞水平。 在这里，我们提出了一个平台，在无细胞合成生物学随机性效应的研究。该微制造平台包含飞升规模的反应C昂贝尔其可以打开（在进出室的自由扩散）和状态封闭（内腔密闭反应物）之间迅速地转换。在闭合状态下，我们限制无细胞蛋白质合成（CFPS）反应表达绿色荧光蛋白（GFP），以及使用时间推移荧光显微镜28（ 图1）遵循基因的表达。我们通过测量随机波动的基因表达的结构中，直接的类似用于表征细胞25的方式表征这种细胞的环境。非微细加工方法的限制无细胞反应包括囊泡和脂质体29-32，水包油乳剂12，和多孔介质33。然而，虽然这些方法可以在密闭的卷34的粒度分布提供控制，微细加工方法创建具有紧紧指定尺寸高度复制的功能，即使在纳米级。此外，这些刚性结构能够容易地随时间跟踪而不易被蒸发或变化的外部环境。在先前的工作中使用8,35不能迅速密封该反应室下列反应开始，当反应开始（时间零）复杂的时间的明确分配微制造容器的设计。用这里介绍的方法中，只有4-5分钟，需要起始和设备上的反应的可视化之间，从而提供了良好定义的“零时间”。以下协议描述的方法用于制造和测试该装置，包括光学光刻，装置组件中，设备测试，并用于图像分析的方法。

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			<td height="20" style="height:20px;">SU-8 2015</td>
			<td>Microchem</td>
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			<td>Toxic. Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td>
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			<td>SU-8 2000 series</td>
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			<td>Microchem</td>
			<td>SU-8 2000 series</td>
			<td>Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td>
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			<td height="20" style="height:20px;">Chlorotrimethylsilane</td>
			<td>Sigma Aldrich</td>
			<td>92360 FLUKA</td>
			<td>Hazardous. Corrosive to the respiratory tract., Reacts violently with water.</td>
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			<td height="20" style="height:20px;">Sylgard 184 PDMS</td>
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			<td>SYLGARD 184</td>
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			<td height="20" style="height:20px;">0.75 mm hole-puncher</td>
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			<td>Harris Uni-Core</td>
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			<td>/NE-231PL-25</td>
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			<td height="20" style="height:20px;">#0 glass coverslip</td>
			<td>Ted Pella Inc.</td>
			<td>260366</td>
			<td>Gold Seal</td>
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			<td height="20" style="height:20px;">Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
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			<td>Nikon Instruments</td>
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			<td height="20" style="height:20px;">CCD camera</td>
			<td>Roper Scientific</td>
			<td>CoolSNAP-HQ</td>
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			<td height="20" style="height:20px;">Shutter</td>
			<td>Shutter Insturment</td>
			<td>Lambda SC</td>
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			<td height="20" style="height:20px;">Light Source</td>
			<td>Nikon</td>
			<td>Intensilight C-HGFI</td>
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			<td height="20" style="height:20px;">Color Filters</td>
			<td>Chroma Technology Corp.</td>
			<td colspan="2">ZET 532/106 excitation, ZT 532rdc dichroic, ET 595/50m emission</td>
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			<td height="20" style="height:20px;">100x oil-immersion objective</td>
			<td>Nikon</td>
			<td>N.A. 1.4</td>
			<td></td>
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			<td height="20" style="height:20px;">Temperature Regulator</td>
			<td>Oko Lab</td>
			<td>H201-T</td>
			<td></td>
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			<td height="20" style="height:20px;">Metamorph</td>
			<td>Universal Imaging Corp.</td>
			<td>Version 7.8.3.0</td>
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			<td height="20" style="height:20px;">Marsh Bellofram transducers</td>
			<td>Marsh Bellofram</td>
			<td>T2000</td>
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			<td height="20" style="height:20px;">24 gauge PTFE tubing</td>
			<td>Component Supply Co.</td>
			<td>/SWTT-24-C</td>
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			<td height="20" style="height:20px;">Septum vials</td>
			<td>National Scientific</td>
			<td>C4015- 17W</td>
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			<td height="20" style="height:20px;">Power Supply</td>
			<td>Hewlett Packard</td>
			<td colspan="2">6205B Dual DC Power Supply</td>
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			<td height="20" style="height:20px;">sharp 23 ga needles</td>
			<td>Precision Glide</td>
			<td>305129</td>
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			<td height="20" style="height:20px;">Male-to-male luer lock adapters</td>
			<td>Qosina</td>
			<td>20024</td>
			<td>Polycarbonate</td>
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			<td height="20" style="height:20px;">Stainless Steel Blunt Needle 23 Ga.</td>
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			<td>/NE-232PL-5C</td>
			<td>Polypropylene</td>
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			<td height="20" style="height:20px;">S30 E coli protein expression system</td>
			<td>Promega</td>
			<td>L1110</td>
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			<td height="20" style="height:20px;">Pet3a-GFP vector/protein</td>
			<td>Novagen</td>
			<td>69418-3</td>
			<td>Assembled in-house. Inserted EGFP gene in Pet3a.</td>
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			<td height="20" style="height:20px;">Quantum Prep Plasmid Midiprep Kit</td>
			<td>Biorad</td>
			<td>#732-6120</td>
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			<td height="20" style="height:20px;">QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
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			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
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			<td height="20" style="height:20px;">Alexafluor 555</td>
			<td>Molecular Probes</td>
			<td>AF555</td>
			<td>http://www.lifetechnologies.com</td>
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		<tr height="20">
			<td height="20" style="height:20px;">ImageJ</td>
			<td>National Institutes of Health</td>
			<td>Version 1.46r</td>
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			<td height="20" style="height:20px;">Plugin: Time Series Analyzer</td>
			<td>Balaji J Dept. of Neurobiology, UCLA</td>
			<td>Version 3.0</td>
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			<td height="20" style="height:20px;">Plugin: StackReg/TurboReg</td>
			<td>Ecole Polytechnique F&#233;d&#233;rale de Lausanne Biomedical Imaging Group</td>
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sealable femtoliter chamber arrays for cell-free biology

无细胞系统可提供用于探测生物反应从复合物资源共享中分离的特定的网络的灵活平台（ 如，全基因表达，细胞分裂）的活细胞内遇到。然而，这样的系统，在传统的宏观尺度散装反应堆使用的，往往不能显示出的动态行为和效率生活微观尺度对应的特征。了解内部的细胞结构和规模上的反应动力学的影响，关键是要理解复杂的基因网络。在这里，我们报告了地限制无细胞反应在细胞水平量，同时使封闭的分子系统的灵活特性一个微型设备。该多层聚（二甲基硅氧烷）（PDMS）器件包含一弹性膜，其可被致动（打开和关闭）飞升规模反应室。当启动时，腔限制无细胞蛋白质合成（CFPS）反应表达荧光蛋白，允许反应动力学随时间使用时间推移荧光显微镜的可视化。在这里，我们证明了如何此装置可用于测量CFPS反应的方式的噪声结构，它是直接类似于用于表征蜂窝系统，从而使得能够使用在蜂窝式系统中使用的噪声生物学技术来表征CFPS基因的电路和它们之间的相互作用与细胞的环境。

此微制造平台的显着优点是在可控弹性“控制阀”，这是独立地以限制CFPS反应（ 图2A）致动的应用程序。当该装置被致动时，该膜腔被压靠在所述载玻片以限制荧光试剂进入反应室（ 图2C）的阵列。为了验证该室可靠地限制通过实验的持续时间的反应中，一个基本的FRAP（荧光恢复后光漂白）试验进行37。荧光团（AF 555）施加到该装置，并且控制阀被致动;使用显微镜的快门光圈，单个井围荧光团分离并单独光漂白（ 图2D）。选定好成为黑暗和亮度没有恢复，直到控制阀减压20分钟60;之后，解除从玻璃室中。此测试验证，这些反应室保持良好的密封在实验的持续时间。 在最佳条件下，表达的容易可视化蛋白（如GFP或萤光素酶）一个CFPS反应表示被施加到该装置在几分钟内可检测的蛋白质。在反应的寿命，蛋白合成中的反应室的内部和外部被成像和通过测量荧光强度的单元中的每个腔室（ 图3A）内的定量。荧光强度，对应于蛋白质浓度，可被映射随着时间的推移对每个反应室（ 图3D）。 基因表达是一个固有的随机过程，介绍波动（噪声），在每一个分子步骤（合成，分解，蛋白质-DNA结合等 ）20。噪音生物学的一个分支专注于基因电路噪声41的证明价值。表达在无细胞系统将具有源自表达的分子机制以及定义的反应容器的边界表面之间的相互作用外在噪声效果。这些外在的效果可能会变得更加明显作为无细胞反应局限于成更小的反应室。执行的多个密闭CFPS反应时间推移成像的能力，然后使能在狭窄的无细胞系统中的仔细分析噪声结构（大小和动力学）的方式直接类似于已报道的用于蜂窝系统25的方法。 图3C和三维示出了从T7启动子组成的GFP表达的时程在一个标准384孔微孔板用孔体积15微升，相比于在PDMS反应室10微米的直径，对应于仅约300 FL卷，约七阶震级第较少。在10微米的反应室蛋白表达率的变化比在所述孔板测量高得多，接近那些见于细胞。 在设备上执行复用的反应表现出相似的动力学上酶标仪（ 图3B），那里是高原，通常假定要由资源限制反应体积42,43内引起迅速增加荧光散装执行CFPS反应。此确定性生长行为，虽然波动，一般是一致的所有的反应室，与实验之间-通过在实验平均腔室之间的痕迹，所述确定性趋势可从跟踪值中减去，离开反应（ 图4A）的只有噪声分量图4B显示了去除确定性，瞬态组件（顶部后GFP表达噪音），以及噪声（底部）的自相关，而图4A示出了在10微米反应室对应的痕迹。在半倍的自相关迹线的分布给出的频率的噪声的依赖性，而自相关迹线的零滞后时间给出噪声的大小，作为方差。 <img alt="图1" src="/files/ftp_upload/52616/52616fig1.jpg" /> 图1.细胞蛋白质的合成反应物被限制在飞升规模的反应室，用于测量基因表达的噪声的目的。从商业无细胞蛋白质表达系统的反应物用于组成型表达绿色荧光蛋白内密闭的PDMS个反应室。这些室的阵列可随时间推移的荧光显微镜，以表征蛋白质的表达和基因表达的噪声被可视化。荧光强度Øf各自反应室随着时间的推移，可以绘制成一个单独的痕迹。 <a href="https://www.jove.com/files/ftp_upload/52616/52616fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52616/52616fig2.jpg" /> 图2.制造与密封飞升规模室两层微流体装置。（A）布局和分解图的设备层。该装置是由两个PDMS层和一个玻璃盖玻片。的PDMS膜，玻璃和控制阀的层之间的密封，保持反应室。的PDMS反应室（B）的SEM图像。内径为10微米。 （C）示意图输入 ​​通道的设备。无细胞蛋白质合成（CFPS）试剂通过反应通道飞行。水被加压，在控制阀来压缩反应室对着载玻片，密封腔室37。从文献转载。 37从化学的英国皇家学会的许可。 （D）荧光漂白恢复（FRAP）在单一使用以及FITC表明室测试后是良好的密封，以防止外部环境。荧光团被捕获在腔室（上图）和单个孔中光漂白（低级图像）。无荧光恢复主要出现在光漂白室，直到控制阀被释放。 <a href="https://www.jove.com/files/ftp_upload/52616/52616fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52616/52616fig3.jpg" /> 图3. EGFP表达的密闭无细胞反应。（A）的密封反应C荧光图像<html>昂贝尔在反应选择的时间点。蛋白生产既能内的反应室，并在主通道的腔室外面可以看到。 （B）中的EGFP克隆到一个Pet3a载体，提供了T7聚合酶启动子和终止子和一个强的核糖体结合位点（RBS）。 （C）的 EGFP的在酶标仪进行堆积无细胞反应的组成型表达的标准化荧光测量。 CFPS反应通常会产生蛋白质放缓至一个“稳定状态”的荧光，然后很快-这是与资源限制43有关。黑色虚线表示的平均跟踪。 （D）从51反应室读了几个实验51原始荧光强度的痕迹归荧光。黑色短线表示平均跟踪在几个实验中，其中示出了蛋白质的表达的确定性分量。/52616/52616fig3large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52616/52616fig4.jpg" /> 图4.个人噪声痕迹物证与细胞和无细胞系统的噪声自相关 。 （a）来自奥斯汀。等 ，2006年中的噪声GFP表达（上图），并从追踪GFP产量活菌25获得归一化自相关函数（下部）。从麦克米伦出 ​​版公司重印许可：[自然] 25（卷439），版权（2006年）。 （B）噪声GFP表达（顶部）和GFP生产无细胞系统获得的归一化自相关函数（底部），跟踪微流体装置反应室。 <a href="https://www.jove.com/files/ftp_upload/52616/52616fig4highres.jpg" target="_blank">请点击这里</a>查看该图的放大版本。

Watch this Scientific Journal Video about Sealable Femtoliter Chamber Arrays for Cell-free Biology at JoVE.com

Sealable Femtoliter Chamber Arrays for Cell-free Biology

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

密封飞升商会阵列的无细胞生物学

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

sealable-femtoliter-chamber-arrays-for-cell-free-biology

Research

JoVE Journal

Bioengineering

9.4K 次观看.  University of Tennessee, Knoxville. A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

视频: Sealable Femtoliter Chamber Arrays for Cell-free Biology

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 ...

科研

生物工程

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

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 ...

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 ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

一锅纯无细胞系统

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.