我们感谢在麻省总医院的DNA核心的约翰Darga制备的DNA筛查图书馆。 PerkinElmer公司的克里斯托弗Chigas为我们WALLAC 1420照度计的宝贵支持。史蒂芬Ciacco的和安捷伦的马丁Thomae规定的布拉沃机器人的支持。我们感谢罗恩·约翰逊和史蒂夫·提图斯在NIH慷慨地提供他们的双荧光素酶发光分析方案。

对于一个实验性的概述，参见图1。 1，准备报告质粒含有α-突触核蛋白启动子 α-突触核蛋白基因（ 图2）的调控元件跨度约10 kb的，从上游NACP（淀粉样肽的非A的β成分）二核苷酸重复序列1,2到2内含子3，我们在包括这个区域的一部分本报记者构造。我们发现，含有内含子2的成长需要特别程序质粒，概述如下。荧光素酶质粒，pGL4.10和pGL4.75（ 图3），可购自Promega公司，并且可以使用常规的分子生物学协议来传播。 <ol><li>使用配方在表1和表2中的引物扩增的2个组分的α-突触核蛋白的启动子在不同的PCR反应。 </li><li> VERIFY PCR产物上使用QIAquick PCR纯化试剂盒（Qiagen），洗脱在50μl的TE液（Tris-EDTA）的琼脂糖凝胶和清洁。存储洗脱0.9kb的片段于-20℃以备将来使用。 </li><li>消化的3.4kb的PCR片段的总体积，以及5微克pGL4.10的，用SacI和XhoI。治疗的载体以小牛肠碱性磷酸酶（Roche）和凝胶纯化使用的PureLink快速凝胶提取试剂盒（Invitrogen）中。 </li><li>用T4 DNA连接酶（NEB）结扎片段和载体。清洗用的PureLink PCR微试剂盒（Invitrogen）将完成的反应，洗脱在10μl的TE缓冲液中。 </li><li>变换2微升的清洗，连接反应成20微升ElectroMAX Stbl4感受态细胞（Invitrogen）中，并根据制造商的说明电穿孔。摇动在30℃培养箱中，在225 rpm离心90分钟。传播2卷上含有100毫克/毫升氨苄青霉素和孵化在30℃下2天。 </li><li>用牙签，选择接种4-6小菌落于2ml结核病（极品肉汤）。在30℃摇床的O / N。种植这些小量制备文化</li><li>从1.5ml每种小量培养使用的PureLink HiPure质粒小量提取试剂盒（Invitrogen）纯化的质粒DNA。 </li><li>消化小量制备用BamHI和electrophorese在琼脂糖凝胶上。正确的克隆应该产生2.8 kb的和4.9 kb的片段。 </li><li>从一个新鲜的LB（卢里亚肉汤）板的正确克隆重新划余下的小量培养和成长在30℃下2天。 </li><li>选择一个小菌落，接种1.5ml的结核病发酵剂培养物。摇O / N为30°C。不要让起子培养成长到饱和。 </li><li>淡化整个发酵剂每150ml预热结核病和动摇在30℃下2天。之前继续到下一个步骤中，使用1.5ml该培养物需额外小量制备和消化来确认克隆中replicati未变异<html>上。 </li><li>从使用的PureLink HiPure质粒大量制备试剂盒（Invitrogen）的余下培养纯化质粒DNA。这中间质粒的预期收益率是每150毫升培养50-150微克。 </li><li>解冻0.9 kb的片段定格在步骤1.2。重复步骤1.3至1.12克隆的0.9 kb的片段到中间质粒，消化，而不是用KpnI和SacI。结扎，变换，选择和成长为maxipreps如上。消化用BamHI应产生5.8 kb的和2.8 kb的片段。这是将被用于在屏幕的质粒。 </li></ol> 2，准备293T细胞中，记者质粒和DNA库的筛选 293T细胞首先被接种到96孔板中，并转染次日。报告质粒和文库的DNA稀释到96孔板中。我们在美国马萨诸塞州总医院（获得的转染级DNA文库板从DNA核心获得=“_blank”&gt; dnacore.mgh.harvard.edu）。这个协议transfects单个库板染293T细胞2相同的板（ 图1），从而2细胞的板应接种每个库板进行筛选。 注：细胞生长介质中无酚红或抗生素，因为这些干扰荧光素酶检测和转染过程中增加毒性，分别为。 <ol><li>每个库板被屏蔽，重悬胰蛋白酶处理的293T细胞以1.5×10 5细胞/ DMEM中含有14％FBS的ml的浓度。倾入无菌容器（爱思进）。 </li><li>将水库，2透明底96孔板（Corning）中，和一箱过滤器枪头（安捷伦）在机器人上甲板。分配100μl的细胞分化为两个板的各孔中，为每孔1.5×10 4个细胞的密度。 </li><li>离心电池板在50×g离心2分钟，使用低斜坡速度，以确保平等贯穿每个孔中的细胞密度。在37°C和10％的CO 2 O / N。 </li><li>淡化萤火虫和海肾报告质粒至5纳克/微升的无血清培养基。在一个15毫升的锥形管结合稀释液中为5:3萤火虫到海肾质粒的比例（体积比）。 </li><li>移液管的组合的质粒到96孔板的各孔中，分配每个库板17微升，加2-10微升过剩，在每孔中。 </li><li>获得的转染级DNA文库板如上述稀释至13.3纳克/微升用无内毒素的水。每孔的最小体积应为28微升。 </li></ol> 3，进行转染在屏幕的第2天，报告质粒和文库的DNA转染到293T细胞中。 <ol><li>对于每个库板，以聚苯乙烯试管混匀107.5微升RT Optifect（Invitrogen公司）的4.3毫升血清的培养基（1:40稀释）。孵育5分钟，在室温和Ñ​​免除44微升（每个库板）到96孔聚苯乙烯V形底平板（Greiner）中的每个孔中。 </li><li>放置板的稀释Optifect，报告质粒（步骤2.5），库的DNA（步骤2.6），和一个盒上的机器人甲板枪头。 </li><li>吸17微升报告质粒，43微升稀释Optifect，和26微升的DNA库中，每次插入抽吸之间的5微升的空气间隙。该文库DNA应该最后吸气，以防止交叉污染。免除提示的内容到一个新的聚苯乙烯V形底板，混合，盖，并预留20分钟，使脂质/ DNA复合物的形成。如果转多个库板，更换枪头和库板，并重复。 </li><li>揭开Optifect / DNA混合物板，并将其放置在机器人上甲板2板293T细胞。使用单一框的枪头，吸80微升的Optifect的：DNA混合物，慢慢地免除40微升到细胞的每块板。如果反fecting多个库板，重复所有Optifect：DNA的板块。覆盖细胞。 </li><li>离心细胞板在1200×g离心30分钟。覆盖，并返回到培养箱中。 </li><li>于室温孵育4-6小时。在此培养后，通过混合12毫升DMEM含10％FBS和10微克/毫升环丙沙星（的CellGro）对于每个转染的库板制备的新鲜培养基。倒入新鲜介质装入无菌水库。 </li><li>将2盒机器人的提示，细胞的单盘，含有新鲜媒体和废物储存到机器人甲板上的水库。从细胞中吸出100μl的媒介，分装到废物储存器部分地填充有70％的乙醇。使用新鲜的秘诀，吸100微升新鲜培养基，慢慢地分配到细胞。重复单元的各个板块。 </li><li>返回板的培养箱中培养48小时。 </li></ol> 4，进行双荧光素酶检测在屏幕的第4天，在萤火虫和海肾萤光素酶测定法进行。 注：双萤光素酶测定，需要顺序加入2测定缓冲液，3X萤火虫测定缓冲液和3X Renilla荧光测定缓冲液中，如表3中描述的萤火虫和海肾萤光素酶不分泌，从而细胞必须测定荧光素酶活性之前裂解。 。萤火虫测定缓冲液中裂解细胞，并提供了底物的萤火虫荧光素酶; Renilla荧光测定缓冲液淬灭萤火虫信号且提供基片的海肾荧光素酶。 <ol><li>每个库板，如表3中所述制备8毫升的3X萤火虫测定缓冲液从储液，并免除82微升到96孔V形底的板的各孔中。 </li><li>每个库板，还准备12 3X Renilla荧光测定缓冲液中，并分配122微升到96孔V形底的板的各孔中。抛开两者的萤火虫和海肾检测缓冲区。 </li><li>放置一个方块的枪头，一个废物储存器，和2板的细胞（来自相同的库板转染）在机器人上甲板。 </li><li>从各板吸60微升媒体并丢弃在废物储存部分地填充有70％的乙醇。盖板及待用。如果转多个库板，重复板的所有集合。 </li><li>放置2盒枪头，3X的萤火虫测定缓冲液中的板，并在机器人上甲板的一组细胞的板。 </li><li>吸出40微升3X萤火虫测定缓冲液中，并将其添加到细胞中的所述第一板，充分混合。切换至新的枪头，然后为第二单元板。把细胞在室温下10分钟，完成裂解。如果转多个库板，更换的提示和电池板的框，然后重复。 </li><li>从每个细胞板在一个光度计，记录为1秒，每孔的每个孔中记录的发光。返回板到机器人甲板。 </li><li>放置2盒枪头，3X的Renilla荧光测定缓冲液中的板，并在机器人上甲板的一组细胞的板。 </li><li>吸出60微升3X Renilla荧光测定缓冲液中，并将其添加到细胞中的所述第一板，充分混合。切换到枪头一个新的框，然后为第二单元板。如果转多个库板，重复单元板的所有集合。 </li><li>从光度计所有板块记录发光，记录每孔1秒如上。丢弃板。 </li></ol> 5。数据分析<ol><li>计算萤火虫： 海肾荧光比（“F：R比率”）由海肾萤光信号的计数除以萤火虫信号的计数每口井。 </li><li>通过将在F计算感应值：每个孔的平均F R的比值：所述板的R比，不包括孔中的最高和最低的25％。 </li><li>跨重复平均感应值为一个单一的转库板。基因诱导或抑制α-突触核蛋白比三褶皱更被认为是“点击”在此屏幕上，并受到进一步的验证和二次筛选。 </li></ol>

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这项工作是由发牌BacMamreagents（美国专利5731182）获得特许权使用费的支持。

α-突触核蛋白有牵连的帕金森氏病（PD）作为路易体4，考虑特殊病征为疾病细胞内的夹杂物的成分。众多的全基因组关联研究已发现，单核苷酸多态性在α- ​​突触核蛋白与风险偶发性帕金森氏5,6,7增加。虽然比散发性PD不太常见，家族性帕金森病也可在α- ​​突触核蛋白8所造成的突变，以及复制和α-突触核蛋白基因9,10,11的三重，这种现象也出现在散发性PD ...

找出关键基因的调控元件，并在他们身上起作用的因素的能力是根本的为众多生物过程的探索。然而，识别，调节基因表达的稀有细胞类型的因素，如特定神经元群体，是具有挑战性的。在这里，我们提出了一个协议，用于识别α-突触核蛋白（SNCA）的新的转录调节，与帕金森氏症有关，表现在多巴胺能神经元在黑质的基因收杆中脑的致密区。我们使用的是高通量，双荧光素酶， 在体外记者屏幕解构α-突触核蛋白表达在293T细胞中实现这一点。 α-突触核蛋白的启动子是第一个克隆到含有萤火虫荧光素酶基因的报告质粒。含有组成型活性的启动子的控制下的海肾荧光素酶的市售质粒作为内部对照。成分股ë记者构建体共转染到293T细胞中微孔板与从DNA表达文库质粒，使得每孔转染了一个单一的文库质粒。 48小时后，对于每一个记者荧光素酶活性被依次使用双辉光测定法测量。在每个孔中的海肾荧光素酶的活性（F：R比率）板基于归一化后的α-突触核蛋白的响应于每个文库的基因的相对表达通过的萤火虫的比率推断。 该协议提供了筛选大量的基因（〜2,500每星期）就其使用最少的人力（1-2人），最低的成本（约300试剂费用为每微孔板检测），以式激活报告基因的能力的能力。神经细胞的基因的转录调节子（ 如 ，α-突触核蛋白），这是很难培养的神经元耐火遗传操作来研究，可以比在此直接功能测定并排侧。我们在包括我们协议的详细方法进行克隆和含有α-突触核蛋白的启动子的质粒的增长，因为我们发现，当用常规方法（见讨论）生长含有该区域的质粒是不稳定的。我们还包括了一种廉价的替代市售的双荧光素酶试剂，用于高通量测定法。这个协议可以很容易地适应目标的感兴趣的其它调控元件，或以任何需要高通量的瞬时转染过程。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>QIAQuick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28106</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink Quick Gel Extraction Kit</td>
 <td>Invitrogen</td>
 <td>K2100-12</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLink PCR Micro Kit</td>
 <td>Invitrogen</td>
 <td>K310250</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Miniprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-03</td>
 <td></td>
 </tr>
 <tr>
 <td>PureLinkHiPure Plasmid Maxiprep Kit</td>
 <td>Invitrogen</td>
 <td>K2100-07</td>
 <td></td>
 </tr>
 <tr>
 <td>Bravo Robot</td>
 <td>Agilent</td>
 <td>-</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips with Filter</td>
 <td>Agilent</td>
 <td>19477-022</td>
 <td></td>
 </tr>
 <tr>
 <td>Robot Pipet Tips without Filter</td>
 <td>Agilent</td>
 <td>19477-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Clear-bottomed 96-well Plates</td>
 <td>Corning</td>
 <td>3610</td>
 <td></td>
 </tr>
 <tr>
 <td>Reservoirs</td>
 <td>Axygen</td>
 <td>RES-SW96-HP-SI</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651101</td>
 <td></td>
 </tr>
 <tr>
 <td>Polypropylene V-bottomed 96-well Plate</td>
 <td>Greiner</td>
 <td>651201</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive Plate Covers</td>
 <td>CryoStuff</td>
 <td>#FS100</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Phusion DNA Polymerase</td>
 <td>New England Biolabs</td>
 <td>M0530L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAC 2002-D6</td>
 <td>Invitrogen</td>
 <td>2002-D6</td>
 <td></td>
 </tr>
 <tr>
 <td>Calf Intestine Alkaline Phosphatase</td>
 <td>Roche</td>
 <td>10713023001</td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>New England Biolabs</td>
 <td>M0202L</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.10</td>
 <td>Promega</td>
 <td>E6651</td>
 <td></td>
 </tr>
 <tr>
 <td>pGL4.74</td>
 <td>Promega</td>
 <td>E6931</td>
 <td></td>
 </tr>
 <tr>
 <td>ElectroMAX Stbl4 Competent Cells</td>
 <td>Invitrogen</td>
 <td>11635-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Subcloning Efficiency DH5&alpha; Competent Cells</td>
 <td>Invitrogen</td>
 <td>18265-017</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM without Phenol Red</td>
 <td>Invitrogen</td>
 <td>31053-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Optifect</td>
 <td>Invitrogen</td>
 <td>12579017</td>
 <td></td>
 </tr>
 <tr>
 <td>Ciprofloxacin</td>
 <td>CellGro</td>
 <td>61-277-RF</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Hydrochloride Powder</td>
 <td>Sigma</td>
 <td>93287</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris-Base Powder </td>
 <td>Sigma</td>
 <td>93286</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100 Pure Liquid</td>
 <td>Fisher</td>
 <td>BP151-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>Coenzyme A</td>
 <td>Nanolight</td>
 <td>309</td>
 <td></td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma</td>
 <td>A6419</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferin</td>
 <td>Invitrogen</td>
 <td>L2912</td>
 <td></td>
 </tr>
 <tr>
 <td>h-CTZ</td>
 <td>Nanolight</td>
 <td>301</td>
 <td></td>
 </tr>
 <tr>
 <td>PTC124</td>
 <td>SelleckBiochemicals</td>
 <td>S6003</td>
 <td></td>
 </tr>
 </tbody>
</table>

high-throughput functional screening using a homemade dual-glow luciferase assay

我们提出了一种快速，廉价的高通量筛选的协议，以确定α-突触核蛋白与帕金森病相关的基因的转录调节。 293T细胞瞬时转染从一个排列的ORF表达文库质粒连同荧光素酶报告质粒，在1个基因的每孔微孔板格式。 48小时，以确定每个基因库后的α-突触核蛋白的转录，归表达从内部控制结构（一hCMV启动子指导海肾萤光素酶）的影响后，萤火虫荧光素酶活性测定。该协议是由封闭在一个生物安全柜台式机器人，它在96孔板进行无菌液体处理提供便利。我们的自动转染协议很容易适应高通量慢病毒库生产或需要三转染的大量独特的文库质粒的共轭等功能筛查方案nction用一套共同辅助质粒。我们还提出一种廉价的和验证的替代市售的双荧光素酶试剂，它采用PTC124，EDTA和焦磷酸盐来抑制萤火虫荧光素酶活性之前， 海肾萤光素酶的测定。使用这些方法，我们筛选7,670人的基因并确定了68监管α-突触核蛋白。这个协议是很容易修改的目标感兴趣的其他基因。

典型的荧光素酶值，F：R比和为一个单一的半板的感应值被显示在图4中注井H2，32倍诱导的命中。基因会引起过度的毒性（例如，井E3），或井转染较差，会产生低的值既萤火虫和海肾萤光素酶，但平均感应值。基因会引起非特异性诱导荧光素酶活性，也许是通过与pGL4骨干相互作用，将导致萤火虫和海肾萤光素酶一样，产生的平均感应值。大的差异在重复之间的感?...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

我们目前确定采用高通量机器人转染和一个自制的双发光荧光素酶检测转录调控因子的快速和廉价的检查方法。该协议迅速产生直接并排端功能数据数以千计的基因，很容易修改针对任何感兴趣的基因。

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

high-throughput-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.4K 次观看.  Massachusetts General Hospital. 我们目前确定采用高通量机器人转染和一个自制的双发光荧光素酶检测转录调控因子的快速和廉价的检查方法。该协议迅速产生直接并排端功能数据数以千计的基因，很容易修改针对任何感兴趣的基因。 

视频: High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Methods Article

使用自制双辉光萤光素酶检测的高通量筛选功能