Name: establishment of a high-throughput setup for screening small molecules that modulate c-di-gmp signaling in pseudomonas aeruginosa
Uploaded: 2016-06-30T14:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa at JoVE.com

We thank the Tolker-Nielsen lab for their generous donation of reporter constructs used to develop the screen. We also thank members of the Ryan laboratory for their helpful comments and critical reading of the manuscript. The work of the authors has been supported in part by grants awarded by the Wellcome Trust (WT100204AIA senior fellowship grant to R.P.R. and 093714/Z/10/Z PhD scholarship to K.N.R.).

注:纯化的c-二GMP报告质粒的克隆和转化过程在别处讨论12。 1.绿色荧光蛋白的生成标记的c-二GMP记者P.假单胞菌 <ol><li>接种5毫升LB培养基的（LB）培养基与P的单个菌落假单胞菌和以200rpm振荡孵育过夜，在37℃。 </li><li>传送2毫升过夜培养物加到200ml新鲜LB培养基在1L烧瓶中，并在37℃以200rpm摇动孵育。 </li><li>在600nm（OD 600）通过从烧瓶中取1毫升样品，并检查使用分光光度计（如前面描述12），每30分钟监测光密度。 </li><li>当OD 600 0.3之间达到- 0.5，放置40毫升培养到50毫升锥形离心管中。 </li><li>通过在4℃以8,000rpm进行10分钟离心沉淀细胞，弃去上清液并且r电子暂停于40ml冰冷，无菌300mM的蔗糖溶液中的细胞。 </li><li>通过如步骤1.5中所述和在20毫升的冰冷无菌，300毫蔗糖溶液再悬浮离心沉淀细胞第二次。 </li><li>通过如步骤1.5中所述，并在400微升冰冷的无菌，300毫蔗糖溶液再悬浮离心沉淀细胞的最终时间。 注意:此时的细胞密度应为每毫升约1×10 11菌落形成单位（CFU / ml）中。 </li><li>冷却在冰上将细胞30分钟，之后，他们准备好电穿孔。 </li><li>添加0.2微克/微升pCdrA :: GFPÇ质粒12溶液，1微升至40微升的P.在1.5 ml离心管，其已经在冰上预冷的上面制备假单胞菌电感受态细胞。混合该悬浮液，并转移到预冷的，及2mm电极间隙，电穿孔杯中。 注:pCdrA :: GFP </em&gt; C:pUCP22Not-P CDRA -RBSII- GFP（Mut3）-T 0 -T 1，放大R通用汽车研究 ，这给了C-二GMP的体育细胞内水平的荧光读出假单胞菌菌株，开发并通过Rybtke 等人 12的验证。 </li><li>上用薄纸的反应杯的外侧除去水分，然后将反应杯放入电穿孔的样品室中。脉冲与2.5千伏，25μF的电容和200Ω电阻的电压。 </li><li>取出试管，加入1 ml LB培养基。然后，将细胞转移到无菌的1.5 ml离心管，并在37℃下以200rpm摇动孵育2小时。 </li><li>蔓延10微升，50微升和培养的100微升等分试样到补充有氨苄青霉素（在100微克/ ml的浓度）无菌的LB琼脂平板上，并在37°C孵育平板过夜。 </li><li>确认GFP表达（excitati关于在485纳米，在520纳米发射）通过与标准的GFP通道在荧光显微镜下检查板，并挑选单菌落来接种5毫升LB.如步骤1.1中所述孵育过夜。混合将0.5ml过夜培养物用在2 ml0.5毫升50％甘油螺杆上管并储存于-80℃。 </li></ol> 2.制备发酵剂培养物接种<ol><li>屏幕前两天，镀P.假单胞菌菌株（含有pCdrA :: GFPÇ质粒）从-80℃的库存到的LB琼脂平板通过用无菌接种在板轻轻扩频细菌（补充有氨苄青霉素100微克/ ml的浓度）循环。过夜孵育所述板在37℃。 </li><li>放映前的晚上，接种P的单个菌落从板到10ml LB培养基的绿脓杆菌在涂（以100微克/毫升的浓度补充有氨苄青霉素）是并以200rpm摇动孵育在37℃下预培养过夜。 </li><li>在屏幕的当天，通过用新鲜LB培养基首先稀释到的OD 1.0 600，然后进一步用5％的LB稀释至OD制备从过夜预培养一个传代培养0.04（1:25比率）600 。 注意:接种培养基的总体积取决于板的数目进行测试。 5％的LB通过稀释100％的LB用磷酸缓冲盐水（PBS），以所需的浓度进行。重要的是，媒体（5％LB）允许pCdrA构激活::在P. GFPÇ 铜绿假单胞菌 。 </li><li>以最低的速度在磁力搅拌器上添加无菌磁力搅拌棒放入容器中，并搅拌所述培养在室温下30分钟，使它们被分配到384孔板之前细菌使其恢复到该介质。 </li></ol>包含384孔板3.接种和孵化小分子注意:不育和良好的无菌技术是最重要下面的步骤。 <ol><li>要准备阳性对照（100％抑制），加入20微升硫酸妥布霉素（25毫克/毫升）在步骤2.3准备的亚文化10毫升（充足长达12板），轻轻混匀。吸移管将40μl阳性对照培养的成孔A23 - P23（ 图2）。 </li><li>以制备阴性对照（0％抑制），添加30微升二甲亚砜（DMSO）中，以在步骤2.3中制备的传代培养中的10ml（足够多达12片），并轻轻混合。吸移管将40μl阴性对照培养的成孔A24 - P24（ 图2）。 </li><li>对于每一个与小分子加盖板的，接种40微升稀释过夜培养物（在步骤2.3中所述）的入井A1的体积- P22（ 图2）。 注意:这可以使用液体处理器抢来实现OT。由于细菌培养物的异质性，重要的是保持连续和适度搅拌以保持细菌始终如一地分布在媒体而分配是重要的。要做到这一点，请从步骤2.4搅拌水土不服文化磁力分配过程中。 </li><li>密封与透气性盖密封所述板，并在37°C孵育6小时。 注意:可透气密封是至关重要的，以保持在所有384个孔不变的条件和横跨板绕过氧气梯度的潜在发展。 </li></ol> 4.生长（OD 600）和细胞内的c-二GMP水平测量（GFP） <ol><li>前约30分钟光度法测量，预暖板读取器至37℃，以避免冷凝。 </li><li>阅读前轻轻地取出透气盖公章。测量在600nm波长下的光密度为每孔10闪烁的设定和0.2秒的稳定时间。 </li><li>之前测量荧光，使一个自动增益和焦点调整基于以及A24（阴性对照），并在75％（ 图2）设置的增益目标值。 <ol><li>从酶标仪控制软件，点击开始测量，然后点击""焦点和增益调节/板标识&#39;标签。 </li><li>选择窗口右侧&#39;调焦&#39;&#39;和&#39;&#39;通道的&#39;&#39;。 </li><li>选择&#39;增益调整&#39;&#39;和&#39;&#39;选择好&#39;&#39;和&#39;&#39;75&#39;&#39;为&#39;&#39;目标值&#39;类型英寸最后，点击&#39;开始调整&#39;前板布局挑好A24。一旦调整完成后，点击&#39;开始测量&#39;。 </li><li>测量从GFP报告荧光在五百二十零分之四百八十五纳米的激发/发射最大值与每孔和一个settli 10闪烁的设置NG时间0.2秒。 </li></ol></li><li>收集审查所有板块的数据。注:数据读数被自动保存在酶标仪控制软件默认。 <ol><li>通过点击"火星人"图标，在控制软件中打开的统计分析软件包查看读数。 </li><li>检索由在感兴趣的盘数"双击"数据访问数据进行分析。 </li></ol></li></ol> 5.数据分析<ol><li>评估使用鲁棒Z&#39;分析，这是报告的高通量筛选中最常见的质量度量测定的均匀性和再现性。强大的计算Z&#39;用以下公式: <img alt="式（1）" src="/files/ftp_upload/54115/54115eq1.jpg" /> 哪里的MAD（平均绝对偏差）是标准偏差的估计值，p是阳性对照（100％抑制），n是阴性对照（0％抑制）为外径600和GFP values​​。注意:用健壮Z&#39;值大于或等于0.5（同时适用于外径600和GFP的原始数据计算出的）的测定被认为是一个极好的测定。 </li><li>计算使用下式生长的抑制％: <img alt="公式（2）" src="/files/ftp_upload/54115/54115eq2.jpg" /> 其中xi OD600是每个样本的迭代OD值600好。 ％抑制OD600&gt; 0，生长抑制剂;抑制％OD600 &lt;0，促生长剂。 </li><li>评估使用式C二GMP的细胞内水平的％抑制（任意荧光强度单位的改变而在细胞密度的变化校正）: <img alt="公式3" src="/files/ftp_upload/54115/54115eq3.jpg" /><img alt="公式4" src="/files/ftp_upload/54115/54115eq4.jpg" /> 其中xi GFP是每个样本的迭代GFP值好。抑制％GFP&gt; 0，C二GMP抑制剂; ％＃160;抑制GFP &lt;0，C二GMP启动。 </li><li>在从中间（中位数±3 MAD），从样品计算3 MAD设置命中检测的阈值以及读数各板的，假定的数据点正态分布。注:但是，±50％抑制截止可以更多的再现和精确的剂量反应测定如果需要选择。 </li></ol>

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The authors have nothing to disclose.

为了改善细菌感染的治疗，很显然，在分子调控水平更好地了解细菌行为是必需的。此处描述的过程将是谁想要发现有操纵或细菌C-二GMP的细胞浓度干扰的可能性小分子微生物学家，生物化学家和临床医生有利。该方法利用一个最近开发的GFP bioreporter并监控在体育的c-二叔GMP细胞水平铜绿假单胞菌 12。此bioreporter已被验证，并且示出重要的不影响使用的培养时，在5％的LB在PBS菌株的生长。使用全细胞屏幕来识别小分子改变体内的c-二叔GMP水平通过细菌膜克服了基于目标的药物发现的主要困难在分子渗透方面，特别是通过那些革兰氏阴性细菌的。更重要的，T他分析显示为持续高于0.5在所有屏幕迄今观察到的一个强大的Z&#39;值非常稳健。使用该协议，就会发现一个数字，抑制和/或促进细胞内P. C-二GMP水平的小分子筛选铜绿假单胞菌 。此外，该测定中也具有识别杀菌或导致在OD 600读出的降低抑菌化合物的潜力。 虽然在协议部分没有讨论，存在用于实验的制备几个​​重要的考虑因素。至关重要的是，请注意该GFP bioreporter是基于质粒来承担。虽然报道质粒被称为是在巴斯德非常稳定假单胞菌 ，它可以连续重新铺板后丢失，因此需要用新鲜镀菌株从-80℃甘油库存，以及检查荧光表达是至关重要的。这也是非常有价值的整个屏幕保持均匀的生长条件因为在这些条件的任何波动有可能在屏幕上的连锁效应。这将包括使某些该介质和抗生素是在批量预制和整个屏幕的过程中使用。细菌培养没有增长（基于OD 600读数）以统一的方式对这种性质的大多数高通量筛选的共同课题。这可能是由于边缘效应或分配不均匀的细菌培养到板中。对于前者，确保透气密封孵育过程中使用是至关重要的。而对于后者来说，吸用培养物的体积的液体处理器管道至少三次建议的配管本身的死体积。它是保持在最小速度磁力搅拌器分配期间的关键。同时监测和用于在实验过程中持续增长的384孔板的存储是极为重要的，这种情况以避免是板的孵育期间的堆叠，因为它可引起非想要氧气梯度导致经济增长方式的倾斜。同样重要的是，以确保用作阴性对照的DMSO车辆没有负面感兴趣的菌株的生长影响。许多与生长相关的这些问题可以通过用之前筛选感兴趣的细菌菌株进行模拟屏幕来避免。数据解释也值得考虑因为C-二GMP抑制的结果是由必须在细胞密度的变化进行修正任意荧光强度单位变化来计算。考虑到这一点不同的数学公式来评估从这些实验的数据输出也应考虑。例如，化合物可以显示为一个C二叔GMP的抑制剂，但实际上可以是一个生长抑制剂，或反之亦然，需要鉴定的命中谨慎解释。 有几个缺点和限制了必须的这种发展和表现过程中被认为是高通量细胞为基础的屏幕。例如，使用荧光探针，其检测性能可能潜在地通过在屏幕中使用的小分子的影响对细胞的c-二叔GMP水平的间接测量生物记者功能。切向的问题是，在基于细胞的测定给出关于在细胞内的c-​​二叔GMP水平的变化的机理没有信息的事实。因此，从实验重要的观察，必须使用高性能液相色谱 - 质谱方法，它被认为是在细菌中测量细胞内的c-​​二叔GMP波动的"金标准"的方法来确认。此外，我们的程序只能提供关于在体外生长的细菌的行为的信息，如细 ​​菌细胞在宿主的上下文生长（体内 ） 快速修改他们的活动，由于这种环境。此外，使用GFP的bioreporter的处理装置的屏幕不采取考虑到细菌细胞的生理状态。然而，该协议可以在屏幕的过程中适应单个孔的微观监控。 即使有这些考虑和限制，这种高通量检测仍然是能够与细胞内的c-​​二GMP水平干扰的小分子一个强大的屏幕。由于许多微生物物种，包括许多细菌病原体已经以表征细胞内的c-​​二叔GMP级别的调制进行了研究，我们的协议可以被应用到不同的细菌种类，并扩大到更复杂的多物种细菌模型的研究。该测定可用于其它细菌种类所使用的生长条件，或者可以适于通过使用不同bioreporters读取其它输出变化容易最优化。不同的孵育时间可以根据所关注的生长阶段施加。但是，扩大或通放增加时，它是importan<html>吨至牢记，细菌还是会在板筹备和读出测量越来越大。因此，建议在使用这种协议的时间为最多15板的屏幕。此外，使用板与超过384孔可能不允许均匀生长，需要进一步优化。当按比例缩小板的数量，这可能是更合适的使用电子移液器代替液体处理机器人手动接种。很明显，这个协议可用于调查该分散生物膜的小分子，因为抗生物化合物干扰用c-二叔GMP的信令。该协议还可以重建，了解细菌生理的各个方面。定将c二叔GMP的信令是在大多数细菌流行，通过使用这个协议中，我们可以识别不同的化学刺激，以确定其工作机制是否需要的c-二叔GMP信令研究这些生物体的生理机能。 _content"&gt;总之，该方法在这个协议提供的鲁棒性和通用性将有助于的c二叔GMP在许多生物系统信令化学调节剂的鉴定。

细菌耐药性的快速发展，对临床上重要的抗生素是目前全球面临的卫生专业人员关心的主要问题之一。传统的抗生素此问题已推动新的搜索可与参与毒力和疾病进展1细菌干扰的过程的化学物质。一个这样的监管系统，利用细胞内第二信使循环二GMP（C-二GMP）最近已成为有希望的有效性2-4的目标。已确定，这种全球第二信使信号分子调控许多功能，包括抗生素抗性，附着力，生物膜形成和疾病2-4。 它现在理解的c-二叔GMP的细菌细胞中的细胞水平由合成和降解，由此GTP两个分子用于通过GGDEF结构域的diguanylate环化酶（DGCS）WH合成的c-二叔GMP的控制ereas的c-二叔GMP降解是由具有要么是EAL或HD-GYP域磷酸二酯酶（PDE）催化（在（3综述，5））。含有这些结构域的蛋白通常包含其它信令域，这表明它们在C二叔GMP周转活性通过3,5-环境或蜂窝线索直接或间接地调节。因此，C-二GMP信令功能，以不同的环境信号的检测细菌的表型链接到修改。 C-二GMP发挥在细菌在转录，转录后和翻译后的通过各种机制4水平的调节作用。 的c-二叔GMP在许多细菌细胞的一个主要影响是在细菌"生活方式"，特别的测定，在附着于表面或组织在生物膜的多细胞结构的能动浮游细胞和柄细胞之间的转换的控制3,5。一般来说，高的蜂窝C-二GMP的水平与生物膜的形成和sessility有关，而低细胞水平上鼓励许多细菌病原体3,5蠕动和毒力因子的合成。因此，C-二叔GMP信令的运作的更详细的知识可以得到的策略用于抑制生物膜形成和毒力在细菌病原体。这是给一个艰巨的任务，大多数细菌基因组编码与GGDEF，EAL和/或HD-GYP域（例如绿脓杆菌有超过40的蛋白质）和多效应6,7大量的蛋白质。 然而，即使有这种复杂性，最近的证据表明，策略操作的c-二叔GMP的信令可以被开发要么防止抗生素耐药菌感染显影或使它们易于被经典抗生素2共同施用的免疫系统或有效的治疗。在与此线，已经实验证明，人工减少细胞内的c-二叔GMP在体外 -grown P的铜绿假单胞菌导致减少生物膜的形成和易感性增加抗菌剂，而P.假单胞菌 -developed上硅胶植入生物膜，位于小鼠的腹膜腔中，可以用类似的方式8-11进行分散。 这里，我们描述了高通量，基于荧光的报告基因检测来筛选小分子可以潜在调节在巴斯德蜂窝的c-二叔GMP水平假单胞菌 （ 图1）。该测定法是基于测量的c-二叔GMP使用先前开发的GFP报告其表达转录链接到c二叔GMP响应CDRA子12细胞水平。这个协议描述的方法在P的报道构建的表达感兴趣假单胞菌菌株，化合物板制备，培养接种到384孔板，生长条件，如我们LL作为关于数据收集，管理和分析（ 图1）的信息。总体而言，该协议将有助于研究人员能够识别潜在的新化合物针对C-二GMP在细菌信号，并用于研究，旨在了解体育的生物学铜绿假单胞菌 。

<table><tbody><tr><td>Lysogeny Broth (Lennox)</td><td>Sigma Aldrich</td><td>L3022-1KG</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S0389-1KG</td><td></td></tr><tr><td>Lysogeny Broth with Agar (Lennox)</td><td>Sigma Aldrich</td><td>L2897-1KG</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Sigma Aldrich</td><td>A0166-25G</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516-1L</td><td></td></tr><tr><td>Phosphate Buffered Saline, pH 7.4</td><td>Life technologies</td><td>10010-056</td><td></td></tr><tr><td>Tobramycin sulfate</td><td>Sigma Aldrich</td><td>T1783-100MG</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>276855-250ML</td><td></td></tr><tr><td>Genesys 10S UV-Vis spectrophotometer</td><td>Thermoscientific</td><td>840-208100</td><td></td></tr><tr><td>2 mm gap electroporator cuvette</td><td>Bio-Rad</td><td>1652092</td><td></td></tr><tr><td>BioRad GenePulser XCell electroporator</td><td>Bio-Rad</td><td>1652662</td><td></td></tr><tr><td>Leica Fluorescent Stereomicroscope</td><td>Leica Microsystems</td><td>MZ16FA</td><td></td></tr><tr><td>BRAND magnetic stirring bar, PTFE, cylindrical with pivot ring (sterilize by autoclaving before use)</td><td>Sigma Aldrich</td><td>Z328952-10EA</td><td></td></tr><tr><td>384-well, white-walled, clear-bottom plates</td><td>Greiner</td><td>781098</td><td></td></tr><tr><td>Multidrop Combi reagent dispenser</td><td>Thermo Scientific</td><td>5840300</td><td></td></tr><tr><td>Multidrop Combi tubing</td><td>Thermo Scientific</td><td>24073290</td><td></td></tr><tr><td>VIAFLO II 16-channel electronic pipette</td><td>Integra Biosciences</td><td>4642</td><td></td></tr><tr><td>100 ml sterile disposable reagent reservoirs</td><td>Fisher Scientific</td><td>12399175</td><td></td></tr><tr><td>AeraSeal air-permeable membranes</td><td>Sigma Aldrich</td><td>MKBQ1886</td><td></td></tr><tr><td>Pherastar plate reader (Software version: 4.00 R4, Firmware version: 1.13)</td><td>BMG Labtech</td><td>Pherastar FS</td><td></td></tr></tbody></table>

establishment of a high-throughput setup for screening small molecules that modulate c-di-gmp signaling in pseudomonas aeruginosa

传统的细菌耐药，带动研究试图在最近发现的监管途径，以确定新的药物靶点。 ，利用细胞内的环二GMP（三二 - GMP）作为第二信使调节系统是其中一类的目标。 C-二GMP是一个行为规范广泛的范围内处理，包括抗生素耐药性，生物膜的形成和毒力的发现几乎所有的细菌信号分子。的C-二GMP如何信号抗生素有抗药性的生物膜发展的控制方面的认识已经提出的方法由此核苷酸或这些信号通路的破坏的细胞浓度的改变可能导致减少生物膜形成或生物膜对抗生素的敏感性增加。我们描述一个简单的高通量bioreporter协议，基于绿色荧光蛋白（GFP），其表达的c二叔GMP的反应性启动子CDRA的控制下，快速筛选小分子与调节在铜绿假单胞菌 （ 绿脓杆菌 ）的c-二叔GMP细胞水平的潜力。这个简单的协议可在48小时内向上的3500化合物筛选并具有适于多种微生物的能力。

此处所描述的方法允许单个研究者3500化合物的平均48小时的时间内有效地并成功筛选。高通量筛选协议的概况被示为图1中的流程图。对于目前的文章中，我们在600μM的终浓度筛选潜在的c-二叔GMP调节化合物一规则的五个标准化合物库。然而，有许多可商购的类似药物的和非药物一样，可以在测定中所使用的化合物组。这些组可以是细菌聚焦化合物或随机池化合物，但每个库会断定分配如集这里筛选，其中有极性官能团和多个立体中心的物理化学性质和特性。在这个协议中，细菌被接种到板连同化合物，抗生素阳性对照和一个DMSO溶媒控制，根据图2中所示的布局; 图3示出了由单个库板例举的初级筛选结果。代表性的OD 600和GFP的原始数据分别示于图3A和3B。说明低到高OD 600 / GFP值的颜色渐变热图，用热水（红色）冷却（绿）色，已应用于井值。通过将三色刻度条件格式从最低的OD横跨在电子表格软件产生的热图600 / GFP读出到最高600 OD / GFP读出。相对于DMSO阴性对照低的OD 600和GFP的值分别对应于生长和c二- GMP水平的抑制，而相对于DMSO阴性对照高值分别对应于生长和c二- GMP水平促进。坚固的Z&#39;为OD 600和GFP AR值È根据在协议（5.1）提供的公式来计算。因为它们都在0.5以上（分别为0.694和0.761），测定质量被认为健壮和进一步分析的数据。生长（OD 600）和细胞内的c-二叔GMP级（GFP）的％抑制通过在协议（5.2，5.3）中提供的公式来计算。代表性数据显示于图4A和4B分别示出。指示低到高的抑制％的彩色梯度热图，用热水（红色）来冷却（绿色）的颜色，已经被应用到阱值。抑制％（OD600 / GFP）从基于主屏幕上单独孔的散点图图5A绘制和5B分别为OD 600和GFP数据。 A±50％的截止（用虚线表示）被选择用于点击检测。在此基础上切断潜在的命中显示为红色圆点。两种类型的感兴趣的化合物的可以更换从检测iscerned。在图5A中鉴定的命中是抑制细菌生长的化合物，而在图5B中鉴定的命中是潜在地具有调节的c-二叔GMP的胞内水平的能力的化合物。两种化合物已被确定为用于该测定代表命中。这些化合物A，一个潜在的生长抑制剂和化合物B，一种潜在的c-二GMP抑制剂。化合物A对应于井F8在图3和4，不得不在增长72.5％的抑制。有在周期性二GMP水平，预计相应的抑制作用。化合物B对应于井K3在图3和4，并有在环状二GMP水平61％的抑制与生长没有变化。选择的命中化合物通过了2mM和两倍稀释系列的最高浓度的10点剂量响应测定法进一步测试。的IC 50值来计算基于所述剂量反应曲线（ 图6A和B）。 <img alt="图1" src="/files/ftp_upload/54115/54115fig1.jpg" /> 图1. 概述:高通量设置为筛选小分子对他们的潜力，在调制 P. C-二GMP的细胞水平 假单胞菌 。此概述显示在该测定中使用的协议的步骤一步表示。 P.的细菌菌落假单胞菌是在LB起子培养物生长过夜。使用试剂分配器，将细胞接种到包含所选择的化合物的384孔板中。将板孵育6小时，在这之后的OD 600和GFP值被 ​​测量。使用这些读数，影响C-二GMP的细胞水平可以被识别的化合物。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig1large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/54115/54115fig2.jpg" /> 。 图 2. 384孔板地图用于小分子的筛选测定法从库（浅蓝色）的化合物等分到孔A1 - P22。的"正控制"（红）组成的50微克/毫升硫酸妥布霉素被加入到孔A23的最终浓度 - P23和"阴性对照"（绿色）含有1％DMSO的被加入到孔A24的终浓度 - P24。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54115/54115fig3.jpg" /> 图3. 代表性的成果一个384孔筛板:R具有代表性的原始数据是OD 600（A）和GFP（B）。渐变色的热图，用热水（红色:OD 600 = 0.65或GFP = 250,000）来冷却（绿色:OD 600 = 0.10或GFP = 40,000）表示从低到高价值的色彩，已经被应用到了很好的值。有OD 600 / GFP读数相对于DMSO阴性对照下水井将趋于红色而具有相对于DMSO对照较高的OD 600 / GFP的读数井将趋向于绿色。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig3large.jpg" target="_blank">请点击这里查看大图版本这个数字。</a> <img alt="图4" src="/files/ftp_upload/54115/54115fig4.jpg" /> 图4 代表结果为计算一个384-孔板的百分比抑制作用。该分析％抑制数据表示为两个的OD 600（A）和GFP（B）读数。渐变色的热图，用热水（红色:OD = 600 -150％或GFP = -20％）来冷却（绿色:OD 600 = 100％或GFP = 100％）的颜色表示，从低到高的抑制值，已施加到阱值。小分子，其中潜在的抑制生长和细胞内的c-二GMP将趋于绿色而小分子，其中潜在的促进生长和细胞内的c-二GMP水平将趋于红色。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig4large.jpg" target="_blank">请点击这里查看大图这一数字。</a> <img alt="图5" src="/files/ftp_upload/54115/54115fig5.jpg" /> 为 获得从每个测试小分子，每个小分子抑制％（OD 600 / GFP）图5代表散点图用点和％抑制分布表示对于每个OD 600（A）和绿色荧光蛋白（B）的读数被示出。 A±50％，截止选择了命中鉴定。潜在的命中以红色突出显示， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54115/54115fig6.jpg" /> 图6. 代表性的成果从剂量-从以前的屏幕识别反应分析两种化合物（潜在的生长抑制剂A和潜在的C-二GMP抑制剂B）在10点剂量-反应实验进一步测试了2最高浓度毫和两倍稀释系列。拟合四参数逻辑函数的数据，得到的158微米和193微米的IC 50值分别<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54115/54115fig6large.jpg" target="_blank">PLE</a>ASE点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa at JoVE.com

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

本文介绍了已经成功地建立筛选小分子的大文库其操纵在绿脓杆菌环状二-GMP的细胞水平的潜在能力，提供了抗菌药物发现和化合物测试一个新的强有力的工具的高通量测定法。

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

establishment-high-throughput-setup-for-screening-small-molecules

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

8.4K 次观看.  University of Dundee. 本文介绍了已经成功地建立筛选小分子的大文库其操纵在绿脓杆菌环状二-GMP的细胞水平的潜在能力，提供了抗菌药物发现和化合物测试一个新的强有力的工具的高通量测定法。 

视频: Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

科研

生物工程

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

免疫与感染

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 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 ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

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.

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 ...

4B 半胱氨酸肽酶自噬抑制剂的高通量筛选分析

Cell-Based Drug Screening for Inhibitors of Autophagy Related 4B Cysteine Peptidase

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

作者聚焦:一种基于荧光素酶的选择性检测方法，用于监测细胞中 ATG4B 27 的活性 

Here, we describe a detailed protocol for the use of a luciferase-based reporter assay in a semi-automated, high-throughput screening format.

基于细胞的自噬相关 4B 半胱氨酸肽酶抑制剂的药物筛选

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins ...

高通量设置的建立筛选小分子能调节C-二GMP信令

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