This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.

1.准备使用的PNP-GESS的宏基因组文库<ol><li>在构造一个E.宏基因组文库大肠杆菌用F粘粒载体使用根据制造商的方案5的fosmid文库生产套件。 </li><li>等分试样在-70℃，这是宏基因组文库细胞源100微升用于存储的库。 注意:在这个库的库存为600nm（OD 600）的波长的测定的样品的光密度为约100。 </li><li>在冰上解冻100微升股票宏基因组文库，并在含有50毫升的Luria-BERTANI（LB）和12.5微克/毫升氯霉素接着37℃温育2小时的500毫升烧瓶中接种。 </li><li>收获在通过离心50ml锥形管中的细胞以1000 xg离心在4℃下20分钟。 </li><li>再次快速重悬沉淀在50ml冰冷却的蒸馏水（DW）和离心机在1000×g离心20分钟，在4℃下。 </li><li> RESuspend在50μl冰冷DW沉淀，用10％（V / V）甘油。使用50微升这种细胞等分的电。 </li><li>放置电感受态细胞50微升和pGESS（E135K）5的DNA（为100ng）的混合物在冰冷电穿孔管和电穿孔（1.8千伏/厘米，25μF）的混合物。 </li><li>迅速加入1毫升代谢产物抑制（SOC）中的超级最佳肉汤，轻轻悬浮细胞。 </li><li>转移细胞成14毫升的圆底管中，使用移液管，在37℃下孵育1小时。 </li><li>涂布在含有12.5微克/毫升氯霉素和50μg/ ml氨苄青霉素的LB一个20×20厘米的正方形板500微升回收的细胞。在30℃下孵育板12小时。 </li><li>刮使用细胞刮菌落并收集细胞进入用冰冷元存储介质的50ml锥形管中。 </li><li>离心机在1000×g离心在4℃下20分钟。重悬在10毫升冰冷的细胞存储介质颗粒达成OD 100 600。 </li><li>等分试样20微升细胞贮存在-70℃。 </li></ol> 2.从宏基因组文库删除误报<ol><li>解冻含冰宏基因组DNA和粘粒pGESS（E135K）股票宏基因组文库细胞（从步骤1.13）。 </li><li>接种于2ml的LB 10微升细胞含有50μg/ ml氨苄青霉素和12.5微克/毫升氯霉素的14毫升圆底管中。在37℃下孵育以200rpm摇动4小时。 </li><li>同时，打开FACS机器上打开默认FACS软件。使用以下设置:喷嘴尖端直径，70微米;前向散射面积（FSC-A）的灵敏度，300 V-数放大;侧散射面积（SSC-A）的灵敏度，350 V-数放大;异硫氰酸荧光区（FITC-A）的灵敏度，450 V-数放大;阈值参数，FSC-A值5。 注:给出的典型设置在材料的表中提到FACS设备。调整为其他的FACS设备的设置。 </li><li>通过加入5微升样品加入到含有1ml的PBS 5毫升圆底管稀释来自步骤2.2的宏基因组文库的细胞。 </li><li>稀释样品库放置到FACS仪器的装货港和点击的FACS软件的收购仪表盘Load按钮。 </li><li>通过点击和控制仪表盘上的流量按钮1500次/秒 - 调整事件的发生率到1000。 </li><li>创建日志缩放FSC-A 与对数比例SSC-A散点图在全球工作表通过单击工具栏上的点图按钮。调整散射门R1到包括使用工具栏上的多边形门按钮单事件（细菌种群）。 </li><li>绘制与细胞计数直方图与对数比例FITC-A在工作表上通过点击直方图按钮。然后，调整从细胞组的电压FITC检查员控制窗口之三设置标签，使得钟形分布的峰值小于10 2 FITC-A的。 </li><li>创建日志缩放FSC-A 对 。日志FITC-A通过点击全球工作表上点图按钮的情节。有关使用该工具栏上的多边形门按钮的情节设置分配门R2使得栅极距离发行总量的10％细胞中心（周围的钟峰+ 5％和-5％之间的细胞形曲线）。 </li><li>放置装有含1.2毫升的LB 50微克/毫升氨苄青霉素和12.5微克/毫升氯霉素的FACS仪器的出口与梳理10 6个细胞同时满足R1和R2栅极的收集管。 </li><li>取下收集管中，帽，并轻轻涡旋。 </li></ol> 3.宏基因组酶的筛选<ol><li>孵育在37℃的分选的细胞（来自步骤2.11）以200rpm振荡培养直至OD 600达到0.5。 </li><li>加入1μl复制感应解决方案，以扩增细胞内的fosmid套数。孵育所述细胞在37℃下另外的3小时，以250rpm摇动。 </li><li>为了制备细胞分选，加入0.5毫升的培养细胞和合适的底物（ 对硝基苯酯，对硝基苯基- β-D-纤维二糖苷，或苯基磷酸酯）成一个14毫升的圆底管中，在终浓度为100μM。 <ol><li>对于对照样品，加入0.5毫升来自步骤3.2的培养细胞的成一个14毫升的圆底管中。添加的PBS的体积相同步骤3.3衬底卷。 </li></ol></li><li>孵育在37℃的两个样品以200rpm摇动3小时。 </li><li>同时，用相同的配置，步骤2.3制备FACS机器。 </li><li>添加5微升细胞（分选）和对照细胞的含有1ml的PBS，分别两个5毫升的圆底管中。 </li><li>将含有控制细胞到T中的管他装载FACS的端口，然后点击Load按钮在FACS软件采集仪表盘。通过仪表盘上的控制流量按钮3000次/秒 - 调整事件的发生率到1000。 </li><li>创建日志缩放FSC-A 与对数比例SSC-A散点图使用的多边形门按钮，点击点图按钮，软件的全球工作表上，并调整散射门R1以包含单事件（细菌种群）工具栏。 </li><li>创建日志缩放FSC-A 与对数比例FITC-A散点图通过单击点图按钮在工作表上并设置分配门R2使用工具栏上的多边形门按钮的情节，这样的不到0.1％ R2的栅极内检测到阴性细胞。 </li><li>与分选样品管更换控制样品管，并调整事件发生率1000 - 通过控制仪表盘上的流量按钮3000次/秒。 </li><li>放置装有0.5的收集管LB毫升的FACS仪器的出口，并整理出10 4个细胞满足R1和R2门两者。 注意:分类标准可以在R2的栅极范围的FITC-A的从顶部0.1％至5％。在这个协议中，顶部1％的细胞被收集为阳性。 </li><li>取下收集管中，帽，并轻轻涡旋。 </li><li>散布0.5毫升收集的样品在含有LB，50微克/毫升氨苄青霉素，12.5微克/毫升氯霉素的2琼脂板（90 25mm培养皿），在37℃下孵育该板过夜。 </li><li>如果有必要，重复步骤3.1-3.14进行了FITC-A富集排序额外回合。 </li></ol> 4.命中选型和​​酶活性的确认<ol><li>流式细胞分析<ol><li>从步骤3.14培养板中，选择出了使用LED照明灯的488纳米波长下观察的殖民地选择器无荧光的殖民地。 </li><li>接种所选择的菌落在14毫升ROUND底含有含2ml LB 50微克/毫升氨苄青霉素和12.5微克/ ml氯霉素，在37℃以200rpm摇动，直到它的外径600达到0.5管。 </li><li>加入2微升复制感应解决方案，以扩增细胞内的fosmid套数。孵育所述细胞在37℃下另外的3小时，以250rpm摇动。 </li><li>准备了14毫升的圆底管中，加入1毫升培养的细胞（步骤4.1.3）的。在100μM的终浓度添加适当的底物（ 对硝基苯基乙酸， 对 -nitrophenyl-β-D-纤维二糖苷，或苯基磷酸酯）。 <ol><li>为对照，将来自步骤4.1.3添加1ml的培养细胞成一个14毫升的圆底管中，加的PBS的体积相同步骤4.1.4基板体积。 </li></ol></li><li>孵育在37℃的两个样品以200rpm摇动3小时。 </li><li>同时，用相同的配置，步骤2.3制备FACS机器。 </li><li>加入5微升控制和基板处理的细胞，以含1毫升的PBS，分别为5毫升的圆底管中。 </li><li>放置FACS装置的装载端口上的含管对照细胞​​，然后点击Load按钮在FACS软件采集仪表盘。调整事件发生率1000 - 使用仪表盘上的流量按钮3000次/秒。 </li><li>点击获取按钮来衡量FITC-A。 </li><li>替换与基板处理的样品管中的对照样品管。调整事件发生率1000 - 使用仪表盘上的流量按钮3000次/秒。 </li><li>点击获取按钮来衡量FITC-A。 </li><li>通过对日志缩放的FITC-A绘制细胞计数的直方图比较两组细胞的荧光。 </li></ol></li><li>其他试验证实酶的活性<ol><li>对于选定的酶考生进一步鉴定，采用标准提取亲提取DNA粘粒从商用提取试剂盒cedures和分析的核苷酸序列，或测试体外酶活6,7。 </li></ol></li></ol>

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The authors have nothing to disclose.

提高生物催化剂的生产效率是生物化工为主业9和宏基因组成功的关键被认为是最好的天然酶源之一。在这个意义上说，它是从那里大多数遗传资源还没有被开发10的宏基因组筛选新酶是必不可少的。几种筛选方法已经开发了使用转录激活11,12直接检测酶的产品，但这些技术需要特定代谢物应答转录系统。另一方面，GESS根据使用的苯酚或PNP标记底物是广泛适用的。虽然大多?...

最近开发的高通量的单细胞分析技术允许新的酶从基于其功能活动1大规模的基因库进行筛选。在单细胞水平，调节转录的蛋白质采用通过检测所生产作为目标酶活性的结果，小分子引发报告基因的表达。一种早期方法涉及从富养产碱 E2使用底物诱导的基因表达筛选（SIGEX）方法，其中基材诱导报告蛋白2的表达的酚降解操纵子的分离。 恶臭假单胞菌 NhaR用于选择苯甲醛脱氢酶3，并从谷氨酸棒杆菌 LysG被用于从不同的突变体文库4新的L-赖氨酸生产菌株的高通量筛选。 先前，遗传酶screeniNG系统（GESS）提出了一个普遍适用的筛选平台5。该系统采用酚系识别二甲调节器，DMPR，P的恶臭 。 DMPR（E135K），和DMPR的突变体，也可以在GESS（PNP-GESS）检测p硝基苯酚（PNP）的使用。在生产酚醛化合物靶酶的存在下，GESS在大肠杆菌大肠杆菌细胞发出的荧光信号，能够使用荧光激活细胞分选仪（FACS）单细胞的快速分离。但宏基因组酶的表达似乎是比传统的重组酶的弱;因此，GESS旨在通过与最佳操作条件5沿调查核糖体结合位点（RBS）和终止子序列的组合以检测与最大灵敏度酚类化合物。 之一的GESS的基本优点是，这种单一的方法理论上允许OV的筛选器超过200种不同类型的BRENDA数据库酶（ 表1对于http:// www.brenda-enzymes.info，2013.7）通过简单地采用不同的基板。它表明，纤维素酶，脂肪酶，和甲基对硫磷水解酶（MPH）可使用的pNP-GESS与对硝基苯基丁酸酯，对硝基苯基- cellotrioside和甲基对硫磷，分别为5的适当底物进行检测。最近，证明了碱性磷酸酶（AP），其是使用的pNP-GESS确定的新酶之一，是在冷适应的海洋宏基因组6找到的第一个不耐热的AP。 这里，筛选过程的细节呈现的pNP-GESS检测三种不同类型的enzymes-脂肪酶，纤维素酶，和碱性磷酸酶的活动-and迅速从宏基因组文库5,6-识别新的候选酶。这些过程包括:宏基因组为PNP-GESS预处理和经营FL流式细胞仪分拣机。而获得的匹配将需要测序进一步鉴定，该协议覆盖的步骤执行到使用流式细胞仪的酶活性的确认的步骤。

<table><tbody><tr><td>CopyControl</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid library production kit&amp;nbsp;</td></tr><tr><td>CopyControl Induction Solution</td><td>Epicentre</td><td>CCIS125</td><td>Fosmid copy induction solution</td></tr><tr><td>EPI300</td><td>Epicentre</td><td>EC300110</td><td>Electrocompetent cell</td></tr><tr><td>pCC1FOS</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid vector</td></tr><tr><td>Gene Pulser Mxcell</td><td>Bio-Rad</td><td></td><td>Electroporation cuvette and electroporate system</td></tr><tr><td>FACSAria III</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry (FACS machine)</td></tr><tr><td>AZ100M</td><td>Nikon</td><td></td><td>Microscope&amp;nbsp;</td></tr><tr><td>UltraSlim&amp;nbsp;</td><td>Maestrogen</td><td></td><td>LED illuminator</td></tr><tr><td>50-ml conical tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>14-ml round-bottom tube&amp;nbsp;</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>5-ml round-bottom tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma-Aldrich</td><td>N7653</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl &amp;beta;-D-cellobioside</td><td>Sigma-Aldrich</td><td>N5759</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butylate</td><td>Sigma-Aldrich</td><td>N9876&amp;nbsp;</td><td>Substrate</td></tr><tr><td>Luria-Bertani (LB)</td><td>BD Difco</td><td>244620</td><td>Tryptone 10 g/L, Yeast extract 5 g/L, Sodium Chloride 10 g/L</td></tr><tr><td>Super Optimal broth (SOB)</td><td>BD Difco</td><td>244310</td><td>Tryptone 20 g/L, Yeast extract 5 g/L, Sodium Chloride 0.5 g/L, Magnesium Sulfate 2.4 g/L, Potassium Chloride 186 mg/L</td></tr><tr><td>Super Optimal broth with Catabolite repression (SOC)</td><td></td><td>SOB, 0.4 % glucose</td><td></td></tr><tr><td>2x Yeast Extract Tryptone (2xYT)</td><td>BD Difco</td><td>244020</td><td>Pancreatic digest of Casein 16 g/L, Yeast extract 10 g/L, Yeast extract 5 g/L</td></tr><tr><td>Cell storage media</td><td></td><td></td><td>2x YT broth, 15% Glycerol, 2% Glucose</td></tr><tr><td>pGESS(E135K)</td><td></td><td></td><td>A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.&lt;br/&gt; See the reference 5 in the manuscript for more details.</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry Software Version 7.0</td></tr><tr><td>PBS</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr></tbody></table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

检查三酚基板按照该协议，以确定在泰安，韩国海洋滩涂沉积物的宏基因组文库的小说宏基因组酶。用于文库构建，平均30 - 40 kb的宏基因组序列插入到F黏粒，这是基于对大肠杆菌大肠杆菌 F因子的复制子和呈现为在细胞中的单一拷贝。注意，F黏粒已被广泛用于构造复杂的基因组文库，由于其稳定传播8。 ...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

multi-enzyme-screening-using-high-throughput-genetic-enzyme-screening

Research

JoVE Journal

Biology

8.7K 次观看.  Korea Research Institute of Bioscience and Biotechnology. This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate). 

视频: Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

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 Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

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 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 ...

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 ...

Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be &#8220;forward&#8221;, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be &#8220;reverse&#8221;, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic screens, including those that depend on either loss-of-function or gain-of-function mutations, which respectively decrease or increase the activity of genes. We then explore general protocols for forward and reverse screens in a popular model organism, the nematode worm. Finally, we highlight how screens are applied in research today, for example to better understand gene interactions that may contribute to neurodegenerative diseases.

Forward and Reverse Genetic Screens

遗传的屏幕

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then ...

教育

JoVE Science Education

高级生物学

遗传学

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Source: Sch&#252;ckel, J., et al. <a href="https://www.jove.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

基于不溶性显色生物质的酶筛选:一种评估植物生物质降解酶降解活性的测定法

Source: Sch&#252;ckel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. ...