这项工作是支持的国家生物医学成像和生物工程授予＃EB003307研究所和美国国家科学基金会职业奖自动对焦

微流控装置的设计使用CorelDRAW或AutoCAD软件 基于PDMS的microvalves设备原理:该设备由三个层次组成:流体层，包含各种尺寸，"控制层"，包含必要的微驱动microvalves流体路径，中间薄PDMS膜，则势必microchambers控制层。休息时，由于合规性和疏水性硅橡胶，膜密封（可逆）反对的席位，因此，商会保持彼此孤立的，没有能量输入。可以采用负压（例如，内部真空）阀打开，使PDMS膜偏转向下，从表面上看，支持两个流体商会之间的墙隔开，从而连接流体路径。从真空到大气压力开关的压力设定，可以实现阀门关闭。 使用CorelDRAW，AutoCAD软件设计的流体层和控制层模式。含有这些设计的口罩被印在通过商业服务（CAD /艺术服务，班登，或）高解析度（8,000至20,000 dpi）的透明度电影（口罩未显示）。 制备硅使用标准的SU - 8光刻的主人<ol><li>标准SU - 8光刻方法被用来创建SU - 8"主人"（SU - 8月到2050年，MicroChem，牛顿，MA）为微层，并在洁净室的阀门控制层（在这个视频没有显示）。 </li><li>为了便于释放，之前的PDMS复制的SU - 8，大师们所接触到一个fluorosilane（（tridecafluoro - 1，1,2,2，tetrahydrooctyl）- 1 -三氯硅烷（TFOCS）），在蒸汽硅烷干燥器罐（不干燥颗粒）连接到真空源。干燥器室必须位于内化学通风柜由于TFOCS蒸气腐蚀性质。 </li><li>放置吸水纸毛巾干燥器内室的一小部分。添加TFOCS下降纸巾和疏散室的空气。应用真空1分钟关闭。关闭真空，使沉积30分钟。存放在密闭容器中以供将来使用的主人。 </li></ol>副本成型硅橡胶的主人<ol><li>流体层和控制层是由硅橡胶成型，从SU - 8大师翻版。 </li><li>彻底搅拌的PDMS预聚物和交联剂（10:1重量比），泡沫在10-15分钟，直至气泡明确干燥器。 </li><li>切成1-2厘米长的片硅胶管。油管根据应用选择合适的大小。我们使用1.1​​4 mm内径的导管，在这里轻松地连接到1 / 16英寸外径管后。 </li><li> Duco公司®水泥使用胶水管上的SU - 8的控制层的主入口区域。小心不要使用太多的胶水，硅胶管是相同的组件的PDMS，将被嵌入到PDMS微流体装置，造成空气和流体紧张的进气口/网点和油管。 </li><li>在我们的设备，进气口地区设计的流体和控制层的口罩，但只到一层的设备（例如，控制层）成型硅胶管入口。要创建流体层的进气口，我们手动删除或穿刺膜覆盖入口地区的几节。因此，对齐和装配后，所有的微（那些携带流动以及那些控制阀）是从设备的顶部访问，使底面平面成像设备，使传统的显微镜阶段。 </li><li>小心地倒入冒泡到PDMS的两个主人，在控制层的主油管周围。泡沫再在干燥器中。完成脱泡后，放入65 ° C的烘箱固化&gt; 1小时。 </li><li>删除固化硅橡胶，涵盖了从烤箱的主人。 </li><li>削减个人的主人（每个主包含三个相同的设备）和剥离设备。 </li><li>取出用针或镊子对入口区域胶水。 </li><li>考虑到洁净室的控制层的PDMS。 </li></ol>薄的PDMS膜制造<ol><li>由于在显示设备的原则，中间层由一个〜12微米厚的硅橡胶膜。 </li><li>混合10:1 WT。由涡旋的PDMS预聚物/正己烷（3:1重量比）固化剂的混合比例。 </li><li>移动到一个干净的房间。 （无尘埃的环境是确保关键的PDMS膜，无缺陷;灰尘颗粒可能会导致膜孔和/或缺陷势必副本模具。） </li><li>将一个硅烷3英寸直径的晶圆一个Solitec飞旋的真空吸盘。晶圆硅烷前PDMS的纺纱，以方便PDMS硅表面释放（fluorosilane衍生物）。容易清洗的塑料薄膜包裹着聚四氟乙烯碗外夹头。 </li><li>免除到使用18号针头（为最大限度地减少气泡）的晶圆PDMS /正己烷混合2-3毫升。 </li><li>设置旋转参数。转速7000 RPM为30秒，产生〜12微米厚的的硅橡胶薄膜在。 </li><li>晶圆温度在85 ° C的一个热点板块治愈的PDMS薄膜4分钟。 </li></ol> PDMS的多层器件的粘接和装配<ol><li>把控制层和一个氧离子室的PDMS膜。打开血浆为30秒（氧压30磅，流速3-5 SCFH，550W）。使接触到的PDMS膜氧活化后立即（在5分钟内）的控制层。经验，系统参数，如氧气的压力，流量，和等离子体功率和治疗时间，根据不同的应用配置。 </li><li>等待5分钟，从晶圆移除控制层与膜。 </li><li>拆下进气口区膜，以便控制和流体层是从通过管子的顶部访问。 </li><li>下一个立体（平面）与流体层控制层（如进气口油管）对齐。由于PDMS到硅橡胶密封，没有永久的粘接。 </li></ol>电脑控制的开放和关闭的PDMS microvalves真空或压力<ol><li>设备对齐和装配后，1 / 16英寸外径（1 / 32英寸ID）聚乙烯管插入1.14 mm内径硅胶进气口和连接的进气口的压力来源或流体水库。 </li><li>打开和关闭阀门，真空和空气压力线，通过两个压力调节器连接到一个微型三通电磁阀阵列的压力控制。 </li><li>电磁阀连接到NI数据采集硬件通过LabVIEW软件控制。 </li><li>设备操作和膜偏转一个彩色CCD摄像头（SPOT放疗，诊断仪器，斯特林高地，心肌梗死）的可视化。 </li></ol>并行的两种不同颜色的染料混合在不同的定义纳升量我们展示了一个平行的混频器，用于存储和精确的分卷纳升的水溶液在不同的混合比例混合允许的操作: <ol><li>流体层包含两个数组microchambers:随着阵列，microchambers的大小减小，从左边开始，从200微米× 400微米至200微米× 40微米; 一个 10是一个500微米× 40微米室是使用数组A中的流体连接; 会议厅10右侧是一组对称的大小的增加商会。数组B室设计，总是等于不同的行中的任何两个相邻商会的添加量。一个0，一个0R和B 10，B 10R被设计成各自控制的解决方案A和B，无需混合。 </li><li>控制层，有两个独立的阀门控制集。阀门{V 1}是用来连接两个各自的进气口的腔阵列，而一个是用来连接两个数组每个商会对第二套阀{V} 。 </li><li>开阀设置{V 1}允许两个染料溶液的流动阵列A和B，分别填补microchambers 。可以实现流量的解决方案，通过手或真空拉控制电磁阀。如果气泡在microchambers形式，更多的解决方案可以推到去除气泡，或者该设备可以留几分钟和气泡就会消失，由于透气性的PDMS的。 </li><li>关闭阀门设置{V 1}两个数组中每个隔离室。 </li><li>打开阀门设置{V}允许在不同的阵列相邻商会之间的混合液体。混合仅约需1-2分钟，以完成这些卷。 </li><li>关闭{V}推流体每个流体商会和商会变形回到原来的形状。由于两流体阵列商会，11个大小不同的设计，生产11种不同的混合比例混合在一个单一的步骤。 </li></ol>集成微流体系统，电脑控制微流体细胞培养灌注我们展示了微流体系统，能够自动灌注到一个单一的细胞培养室的多种解决方案。 "入口控制microvalves，可以在任何单一的入口，各种不同的组合，或一次序列激活。该设备是能够生产各种解决方案的梯度或混合物。 该器件还包​​括三个层次:一个流体层，控制层和中间薄PDMS膜。 此设备的替代制作步骤: <ol><li>流体通道和控制通道的入口是"一拳"使用直径1.2毫米微冲哈里斯（特德佩拉公司）。使用减弱了18针插入通过控制层的PDMS针管连接进气口。这允许比硅胶管的进气口密集的包装。遵守的PDMS提供了一个有效地传递流体或气压针周围的密封。 </li><li>如前所述，薄PDMS膜的结合，以控制层是使用接触氧气等离子来完成。 </li><li>流体层副本成型准备与PDMS预聚物，交联剂，在5:1的比例，部分为25分钟在60℃，在对流烘箱固化。在这一点上，部分治愈的流体层仍然是俗气，但它可以从主删除。 </li><li>流体层是手动对齐预装配的控制和使用膜层立体。组装的设备是一个烤盘上，然后放置5分钟，在80 º C下，阀门控制线连接到自动控制器和阀门被启动，直到流体真空应用在所有的阀座层膜分离。 "分离"的阀门后，电脑控制器设置为周期的阀门开启和关闭，而该设备是在80℃的烤盘上进一步治愈至少1个小时。 </li></ol>我们集成的微流体系统的特点:该设备能够自动灌注16不同的解决方案，以细胞培养室，使用复油液计划。渠道设计确保所有的进气口的阻力是平衡的。我们的微型阀的设计分离的解决方案和控制，通过流体迅速清除，这就限制了交叉污染的综合频道漂洗。综合人字形混频器可以被激活，产生不同的进气口的混合物。此外，还有四个不同的电阻，可以激活改变流量的渠道。

<ol>
	<li>Li, N., Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+mixing+of+photolithographically-defined+nanoliter+volumes+using+elastomeric+microvalve+arrays.">Parallel mixing of photolithographically-defined nanoliter volumes using elastomeric microvalve arrays.</a> Electrophoresis. 26 (19), 3858-3864 (2005).</li><li>Thorsen, T., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration.">Microfluidic large-scale integration.</a> Science. 298 (5593), 580-584 (2002).</li></ol>

我们的微阀的设计的主要优点: <ol><li>没有额外的能量来源是需要关闭的流体路径，因此加载的设备是高度可移植; </li><li>该设备可内置光刻图案的SU - 8模具的PDMS副本，允许microfabricating深（可达1毫米），垂直侧壁的渠道（即高度的功能，可以指定独立的宽度），并在很精确的特点。 </li></ol>并行混频器的优点: <ol><li>这是很容易制造和简单控制。 </li><li>卷光刻定义，因此，非常精确。 </li><li>液体和试剂，...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Clean silicon wafers</td>
<td>Supplies</td>
<td>Silicon Sense Inc.</td>
<td>3P0110TEST</td>
<td>3-inch diameter, P/Boron</td>
</tr>
<tr>
<td>"Master" wafers containing SU-8 patterns</td>
<td>Supplies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Fabricated in house using standard photolithography procedures </td>
</tr>
<tr>
<td>Desiccators (2)</td>
<td>Equipment</td>
<td>VWR</td>
<td>24987-048</td>
<td>One for silanization, one for PDMS de-bubbling.</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>OHAUS Corp.</td>
<td>SC6010</td>
<td>&nbsp;</td>
<tr>
<td>Oven</td>
<td>Equipment</td>
<td>Sheldon Mfg. Inc.</td>
<td>1330GM</td>
<td>&nbsp;</td>
<tr>
<td>MiniVortexer</td>
<td>Equipment</td>
<td>VWR</td>
<td>58816-121</td>
<td>&nbsp;</td>
<tr>
<td>Spinner</td>
<td>Equipment</td>
<td>Headway Research Inc.</td>
<td>PWM32</td>
<td>&nbsp;</td>
<tr>
<td>Plasma etcher</td>
<td>Equipment</td>
<td>Plasmatic Systems Inc. </td>
<td>Plasma Preen II-973</td>
<td>&nbsp;</td>
<tr>
<td>Hot Plate</td>
<td>Equipment</td>
<td>Torre Pines Scientific</td>
<td>HP30A</td>
<td>&nbsp;</td>
<tr>
<td>Stereoscope</td>
<td>Microscope</td>
<td>Nikon</td>
<td>TMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>Equipment</td>
<td>Diagnostic Instruments</td>
<td>SPOT RT</td>
<td>&nbsp;</td>
<tr>
<td>Solenoid valves</td>
<td>Equipment</td>
<td>Lee Company</td>
<td>LHDA0511111H</td>
<td>&nbsp;</td>
<tr>
<td>Data acquisition board</td>
<td>Hardware</td>
<td>National Instruments</td>
<td>PCI 6025E, CB-50LP</td>
<td>&nbsp;</td>
<tr>
<td>LabView</td>
<td>Software</td>
<td>National Instruments</td>
<td>Version 8.0</td>
<td>&nbsp;</td>
<tr>
<td>Tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1-trichlorosilane</td>
<td>Reagent</td>
<td>United Chemical Technologies</td>
<td>T2492</td>
<td>Silanization must be done in a chemical fume hood.</td>
</tr>
<tr>
<td>PDMS prepolymer and crosslinker</td>
<td>Reagent</td>
<td>Dow-Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Hexane</td>
<td>Reagent</td>
<td>EMD	</td>
<td>HX0295-6	</td>
<td>

</td>
</tr>
<tr>
<td>Color Dyes</td>
<td>Reagent</td>
<td>Spectrum Chemical Mfg. Corp.</td>
<td>FD&C 110, 135, 150</td>
<td>Blue #1, Yellow #5, Red #3. 
</td>
</tr>
<tr>
<td>3 ml disposable transfer pipets</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>13-711-20</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>Supplies</td>
<td>Kimberly-Clark</td>
<td>34155</td>
<td>



</td>
</tr>
<tr>
<td>Weighing boats</td>
<td>Supplies</td>
<td>VWR </td>
<td>12577-027</td>
<td>




</td>
</tr>
<tr>
<td>Tongue depressor</td>
<td>Supplies	</td>
<td>Fisher Scientific	</td>
<td>11-700-555	</td>
<td>&nbsp;</td>
<tr>
<td>P100 dishes</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>08-772E</td>
<td>		</td>
</tr>
<tr>
<td>Silicone tubing (1.14 mm inner diameter (I.D.))</td>
<td>Supplies	</td>
<td>Cole-Palmer Instrument Co.</td>
<td>07625-30</td>
<td>&nbsp;</td>
<tr>
<td>Tygon tubing (O.D. 1/16 in; I.D. 1/32 in)</td>
<td>Supplies	</td>
<td>Cole-Palmer Instrument Co.	</td>
<td>06418-02</td>
<td>&nbsp;</td>
<tr>
<td>Duco Cement</td>
<td>Supplies</td>
<td>Devcon</td>
<td>6245</td>
<td>



</td>
</tr>
<tr>
<td>Razor blade</td>
<td>Tools</td>
<td>VWR</td>
<td>55411-050</td>
<td>



</td>
</tr>
<tr>
<td>Needles</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>0053482 (25 Gauge)</td>
<td>




</td>
</tr>
<tr>
<td>#5 Forceps</td>
<td>Tools</td>
<td>Fine Science Tools</td>
<td>11251-20</td>
<td>



</td>
</tr>
<tr>
<td>50 ml centrifuge tube</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>05-526B</td>
<td>




</td>
</tr>
<tr>
<td>Seal wrap film</td>
<td>Supplies	</td>
<td>AEP Industries Inc.	</td>
<td>0153877</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microcentrifuge tubes</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>05-406-16</td>
<td>




</td>
</tr>
<tr>
<td>15 ml centrifuge tubes	</td>
<td>Supplies	</td>
<td>BD Falcon	</td>
<td>352097</td>
<td>&nbsp;</td>
<tr>
<td>Purple nitrile power-free gloves</td>
<td>Supplies 	</td>
<td>VWR 	</td>
<td>40101-348</td>
<td>&nbsp;</td>
<tr>
<td>1.2 mm Harris biopsy punch	</td>
<td>Tools</td>
<td>Ted Pella, Inc.</td>
<td>15074	</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

microfluidic chips controlled with elastomeric microvalve arrays

微型微流体系统提供低成本的护理诊断和高通量的生物医学检测的简单而有效的解决方案。强大的流量控制和精确的流体体积，这些应用程序的两个关键要求。我们已经开发出具有弹性的聚二甲基硅氧烷（PDMS）的微型阀阵列的微流体芯片:1）不需要额外的能量来源，关闭流体的路径，从而加载的设备是高度可移植; 2）允许microfabricating深（可达1毫米）通道与垂直侧壁，在非常精确的特点。 的PDMS microvalves为基础的设备包括三个层次:一个流体层，包含流体的路径和各种尺寸，包含必要的微驱动microvalves流体路径，控制层和中间细的控制，则势必PDMS膜microchambers层。流体层和控制层是由硅橡胶成型副本从SU - 8胶的主人，而薄的PDMS膜是在指定的高度旋转的PDMS。控制层是保税PDMS两个氧活化后的薄膜，然后再组装与流体层。 microvalves是休息时关闭，并可以通过运用负压（例如，内部真空）打开。微型阀关闭和开放的自动化，通过计算机软件控制的电磁阀。 在这里，我们展示了两个不同的应用程序为基础的微阀微流体芯片。第一个芯片可以存储在不同的混合比例混合的精确分卷纳升的水溶液。第二个芯片允许计算机控制的微流体细胞培养灌注。 该设备是编造容易和简单的控制。由于硅橡胶的生物相容性，这些微型芯片可以有微型诊断方法以及基本的细胞生物学研究中的广泛应用。

Watch this Scientific Journal Video about Microfluidic Chips Controlled with Elastomeric Microvalve Arrays at JoVE.com

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

我们展示了用于制造和自动化弹性聚二甲基硅氧烷（PDMS）为基础的，并不需要额外的能量的微型阀阵列关闭和功能光刻定义的精确卷的协议。一个平行subnanoliter量混频器和一个集成微流体灌注系统。

微流控芯片控制，弹性微阀阵列

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

microfluidic-chips-controlled-with-elastomeric-microvalve-arrays

Research

JoVE Journal

Biology

20.9K 次观看.  University of Washington. 我们展示了用于制造和自动化弹性聚二甲基硅氧烷（PDMS）为基础的，并不需要额外的能量的微型阀阵列关闭和功能光刻定义的精确卷的协议。一个平行subnanoliter量混频器和一个集成微流体灌注系统。

视频: Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

制备热塑性微流体通道

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

微流控一次性诊断的应用

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

复用的恒化器阵列的设计与应用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

核小体阵列从重组核心组蛋白和核小体定位的DNA组装

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...