We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

注：吞吐量这种检测系统取决于可用的HTS实验室设备。该协议是由在Tecan自由EVO 200机器人，它在96孔格式进行液体处理容易。小型化到384孔格式也是可能的。机器人液体处理系统是为了在所有的实验步骤保持在无菌条件下放置在层流罩。如果没有液体处理自动化是可用的，该协议可以很容易地适合于低吞吐量的格式。 初筛 1.第一天：准备和种子细胞注：种子细胞，得到80％汇合在测定荧光素酶的活性，在接种后，即 ，48小时的时间。 <ol><li>重悬胰蛋白酶CHP134-mycn3UTR神经母细胞瘤细 ​​胞的2×10 5个细胞/ ml在RPMI含有10％FBS和1％L-谷氨酰胺的浓度。为27片制备至少250毫升<html>细胞悬液考虑到液体处理，波谷死体积和反复移液步骤。倒入细胞悬浮到无菌的贮存器并将其放置在所述机器人处。 </li><li>放置9条形码白色平底96孔板和一个盒上的机器人台200微升无菌吸管提示。各分注步骤之前混合细胞悬浮液通过移液以避免细胞在重力下沉降。分配75微升细胞成9板各孔，以得到每1.5×10 4个细胞的最终密度良好。 </li><li>盖上盖子的板和离开他们的保育箱的外面30分钟以使细胞沉降到井的底部。这将最大限度地减少边缘效应。 </li><li>重复，以制备27片的总数量的步骤1.2和1.3的两倍。 </li><li>将板在37℃和5％CO 2孵育24小时。 </li></ol> 2.第2天：治疗与库化合物的细胞ntent“&gt;注意：频谱收集小分子文库包含2000化合物在10毫在DMSO中的浓度设置在8行和10列的25个96孔板上的第一个和最后列中的孔是空的控件。 。化合物筛选试验一般进行的1-10μM化合物浓度8。这里所描述的筛查方案修复2μM的工作浓度和24小时的试验结束点。 <ol><li>解冻9化合物文库平板在室温下。 </li><li>准备两槽用无菌PBS及PBS-0.5％DMSO的解决方案，并将其放置在机器人的办公桌。每个库板，准备和标记相应一个稀释盘 - 聚丙烯U型底96孔板具有至少400微升一个工作容积。填写列2的每孔稀释板的398微升无菌PBS的使用液体处理器的固定秘诀11。填列1和12用50μl无菌PBS在为了再生在对照孔中的车辆的浓度（0.02％）0.5％的DMSO。 </li><li>将两个库板的相应标记稀释板，两箱50微升无菌枪头和机器人桌子六个单元板。揭开细胞板。 </li><li>吸取2微升相应的稀释板10毫米原液从两个库板块之一拌匀。这将导致在50微米的中间化合物的浓度。使用同一套技巧，取3微升50μM稀释3条形码白96孔板的细胞。这种稀释方案导致2μM工作的每个浓度测试化合物。 注：为避免不必要的操作与技巧，使用全套96提示从一开始，尽管库板仅包含80化合物。这是可能的，因为在第一和库板的最后一栏是空的。 </li><li>更换枪头，重复圣EP 2.4的第二个库板。 </li><li>覆盖细胞板，并将它们返回到培养箱24小时。 </li><li>对于其余的库板重复步骤2.3-2.6两次以上。 </li></ol> 3.第3天：执行萤光素酶检测注：在进行萤光素酶测定法中，也能够与基于刃天青还原，以便从每个孔9得到细胞存活率索引的细胞生存力测定法复它。复用萤光素酶和活力测定测定结果减少荧光素酶信号的，但是，可以忽略，如果细胞提供足够高的水平的萤光素酶的活性。荧光素酶活性可以通过各种商业和自制10,11-萤光素酶测定试剂稳定发光信号来检测。 <ol><li>平衡所选择的重组荧光素酶试剂至室温。确保音量足以让所有的检测板。 27板s170毫升萤光素酶试剂将需要考虑到液体处理，波谷死体积和重复的移液步骤。倾萤光素酶试剂成贮存，并将其放置在机器人处。 </li><li>从孵化器中取出9板的细胞，将它们放在机器人的办公桌，发现，等到平衡至室温。添加50微升每孔平板的荧光素酶试剂的由板。在板之间，引入一个延迟等于一个板的发光读数时间。这将确保所有的板被从试剂加成相等的时间间隔内进行测量。 </li><li> 30分钟温育后，将第一板中的光度计，测量短暂摇动步骤之后发光。重复所有板块。记录每盘的条形码，并将其关联到相应的数据文件，以避免来自大量处理板所产生的错误。 </li><li>重复两次，其余18板与细胞步骤3.2-3.3。 </LI&gt; <li>收集剩余的荧光素酶试剂，并将其储存在-20℃。 </li></ol> 4.第4天：数据分析<ol><li>过程实验数据使用所有样品的归一化，以对板载体处理对照的平均值。对于每个库化合物计算平均值X和SD超过一式三份。 </li><li>选择上调和下调通过设定X的阈值±K＆times SD，其中，平均值X和SD被计算在所有测定处理后的值，而k的命中是一个定义的常量。 </li><li>选择任意的阈值切断&lt;时减少荧光素酶信号的最有可能与化合物毒性相关媒介物处理的对照的20％。丢弃低于该阈值的命中将显著降低了一些在反筛选假阳性命中。 注：代替控制为基础的正常化，实验数据可被处理施加Z分数或其健壮类似物B·S芯12。此外，可用12命中选拔替代的统计方法。 </li></ol> 5.反筛查注意：被确认和评价的特异性通过反筛选在初级筛选中鉴定的化合物（标记为“命中”）。 <ol><li>樱桃采摘从存储在单个2D条码管的等分所选命中，创造新的“打板”，节省了相应的布局。不要忘记留下自由位置的控制。 注：在没有一个有效的樱桃采摘系统，一种可能测试对照和3&#39;非编码区承载细胞并联在初级筛选。在这种情况下，特定​​的选择与只依赖于引入的3&#39;非编码区作用的化合物将初筛消除反筛选的需要之后是可能的。然而，在两种细胞系的平行筛选将增加一倍的检测成本。 </li><li>继协议初筛（第“1天：准备和种子细胞”），为每一个“打板”准备三个96孔板每个CHP134-mycn3UTR和CHP134-CTRL稳定的细胞。 </li><li>继协议初筛（第“2日：治疗用化合物库的细胞”），使用每个打板治疗3 CHP134-mycn3UTR三CHP134-CTRL板在2μM工作浓度。 注意：尽管此处所描述的反画面中一式三份以2微米的单次剂量进行的，这可能是有益的替代标准重复和在3种浓度测试命中10倍稀释液从2微米至20纳米。应用这种方法，不仅允许以确认主画面的数据，而且还以获得在主命中初步剂量​​ - 反应数据，最小化假阳性率。在这种情况下，不同的分析管道应该是APPLI编处理实验数据和确认命中。 </li><li>继协议初筛（第“3日：执行萤光素酶检测”），测量所有CHP134-mycn3UTR和CHP134-CTRL板荧光素酶的活性。 </li><li>下列的协议初筛（参见“第4天：数据分析”），从规格化处理的孔的实验数据，以对板媒介物处理的对照的平均，并计算平均值和SD上重复。对于所测试的各化合物，计算荧光素酶活性在CHP134-mycn3UTR的倍数变化与CHP134-CTRL细胞。选择任意倍数变化下调取舍的&gt; &lt;0.01 1.5和意义p值。 注：在所描述的测定法设置命中特异性的决定因素参数是萤光素酶活性的一个显著倍数变化在CHP134-mycn3UTR与CHP134-CTRL细胞。如果需要的话，我们也可以通过在本身同时进行细胞活力测定评估化合物的毒性lected化合物。这可能是特别有益的，如果初级命中计数器筛选以剂量响应分析。对于单剂量，我们的经验表明减少荧光素酶活性很强的相关性与细胞活力下降6。因此，在靶细胞以及对照细胞减少荧光素酶活性可以被认为是化合物的毒性，在所测试的浓度的指标。 </li></ol>

<ol>
	<li>Schwanh&#228;usser, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Tebaldi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+uncoupling+between+transcriptome+and+translatome+variations+after+a+stimulus+in+mammalian+cells.">Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells.</a> BMC Genomics. 13 (1), 220 (2012).</li><li>Mitchell, S. F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+properties+of+eukaryotic+mRNPs.">Principles and properties of eukaryotic mRNPs.</a> Mol. Cell. 54 (4), 547-558 (2014).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nat. Rev. Genet. 15 (5), 293-306 (2014).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3&#8217;+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3&#8217; untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biol. 9 (5), 563-576 (2012).</li><li>Sidarovich, V., Adami, V., Quattrone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cell-Based+High-Throughput+Screen+Addressing+3&#8217;+UTR-Dependent+Regulation+of+the+MYCN+Gene.">A Cell-Based High-Throughput Screen Addressing 3&#8217; UTR-Dependent Regulation of the MYCN Gene.</a> Mol. Biotechnol. 56 (7), 631-643 (2014).</li><li>Cheung, N. -. K. V., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblastoma:+developmental+biology,+cancer+genomics+and+immunotherapy.">Neuroblastoma: developmental biology, cancer genomics and immunotherapy.</a> Nat. Rev. Cancer. 13 (6), 397-411 (2013).</li><li>Hughes, J. P., Rees, S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+early+drug+discovery.">Principles of early drug discovery.</a> Br. J. Pharmacol. 162 (6), 1239-1249 (2011).</li><li>Herbst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexing+a+high-throughput+liability+assay+to+leverage+efficiencies.">Multiplexing a high-throughput liability assay to leverage efficiencies.</a> Assay Drug Dev. Techn. 7 (3), 294-303 (2009).</li><li>van Olst, E. S. i. e. b. r. i. n. g. -., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affordable+luciferase+reporter+assay+for+cell-based+high-throughput+screening.">Affordable luciferase reporter assay for cell-based high-throughput screening.</a> J. Biomol. Screen. 18 (4), 453-461 (2013).</li><li>Baker, J. M., Boyce, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+functional+screening+using+a+homemade+dual-glow+luciferase+assay.">High-throughput functional screening using a homemade dual-glow luciferase assay.</a> J. Vis. Exp. (88), (2014).</li><li>Malo, N., Hanley, J. A., Cerquozzi, S., Pelletier, J., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+practice+in+high-throughput+screening+data+analysis.">Statistical practice in high-throughput screening data analysis.</a> Nat. Biotechnol. 24 (2), 167-175 (2006).</li><li>Hitti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+ribosomal+protein+promoter-based+reporter+system+for+selective+assessment+of+RNA+stability+and+post-transcriptional+control.">A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control.</a> RNA. 16 (6), 1245-1255 (2010).</li><li>Oikonomou, P., Goodarzi, H., Tavazoie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+regulatory+elements+in+conserved+3&#8217;+UTRs+of+human+transcripts.">Systematic identification of regulatory elements in conserved 3&#8217; UTRs of human transcripts.</a> Cell Rep. 7 (1), 281-292 (2014).</li><li>Gandin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J. Vis. Exp. (87), (2014).</li><li>Conne, B., Stutz, A., Vassalli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3&#8217;+untranslated+region+of+messenger+RNA:+A+molecular+&#8220;hotspot&#8221;+for+pathology.">The 3&#8217; untranslated region of messenger RNA: A molecular &#8220;hotspot&#8221; for pathology.</a> Nat. Med. 6 (6), 637-641 (2000).</li><li>Cheneval, D., Kastelic, T., Fuerst, P., Parker, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+methods+to+monitor+the+modulation+of+mRNA+stability:+a+novel+approach+to+drug+discovery+and+therapeutic+intervention.">A review of methods to monitor the modulation of mRNA stability: a novel approach to drug discovery and therapeutic intervention.</a> J. Biomol. Screen. 15 (6), 609-622 (2010).</li><li>Pascale, A., Govoni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+world+of+post-transcriptional+mechanisms:+is+their+deregulation+a+common+link+for+diseases?+Focus+on+ELAV-like+RNA-binding+proteins.">The complex world of post-transcriptional mechanisms: is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins.</a> Cell. Mol. Life Sci. 69 (4), 501-517 (2012).</li><li>Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+pre-mRNA+processing+and+altered+architecture+of+3&#8217;+untranslated+regions+contribute+to+the+development+of+human+disorders.">Impaired pre-mRNA processing and altered architecture of 3&#8217; untranslated regions contribute to the development of human disorders.</a> Int. J. Mol. Sci. 14 (8), 15681-15694 (2013).</li><li>Bhattacharyya, A., Trotta, C. R., Peltz, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+GEMS--a+novel+platform+technology+targeting+post-transcriptional+control+mechanisms.">Mining the GEMS--a novel platform technology targeting post-transcriptional control mechanisms.</a> Drug Discov. Today. 12 (13-14), 553-560 (2007).</li><li>Peltz, S. W., Welch, E. M., Trotta, C. R., Davis, T., Jacobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+post-transcriptional+control+for+drug+discovery.">Targeting post-transcriptional control for drug discovery.</a> RNA Biol. 6 (3), 329-334 (2009).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

对于基因表达的很长一段转录调控被认为发挥了重大的，如果在控制蛋白质的生产不是唯一的作用。越来越多的证据，但是，表明转录后调控贡献之多，如果不超过，转录调控，以确定细胞蛋白质丰度1,2。基因表达的转录后调控更加复杂和精细的比起初的想法。事实上，转录后调控的所有各个阶段都出现了调控，包括mRNA加工，本地化，营业额，翻译3以及新描述的可逆RNA甲基化4。从一些转录后调控过程可能受到影响，测定下面描述着重于那些涉及mRNA的3&#39;非翻译区（3&#39;非编码区）。 3&#39;UTR广泛影响mRNA的命运通过特定的RNA结合公关互动为主oteins和非编码RNA，以它的调节序列和/或二级结构5。因此，小分子改变这些相互作​​用或干扰的上游信号转导途径将转向，调节蛋白质丰度的平衡。这种治疗方法是用于人类疾病的量存在证据表明疾病相关基因进行转录后调节，从而代表了药物干预的潜在目标特别重要的。因此，系统通过用在高通量形式的转录后控制机制的干扰，允许mRNA水平“调节剂的筛选可成为潜在的新的治疗方法的鉴定有价值的工具。 所描述的基于细胞的报道基因测定允许对能够经由依赖于它的3&#39;端非编码区的机制来调节的mRNA命运的化合物的鉴定。它由几个主要的Components（ 图1），并且需要几个预备步骤来验证该测定的可行性。首先，所述报道构建体被设计为包括3&#39;从感​​兴趣的基因的UTR。的3&#39;端非编码区融合到报道基因的末端，如萤火虫荧光素酶（ 图1）。通过插入的3&#39;端非编码区施加的任何变化在转录后控制机制将改变该嵌合转录物的稳定性和/或翻译效率，从而导致不​​同的荧光素酶的水平。因此，改变荧光素酶活性的服务感兴趣的基因的受影响的转录后调控的间接测量。该系统的第二组分是控制报道构建，表达相同的报道基因，但不包含任何3&#39;非编码区相同的表达质粒。两个报告质粒可以设计在实验室使用常规的分子生物学的协议或从商业来源购买。合适的细胞系的选择是用于测定施工的关键。其中细胞系是最佳的模式取决于很多因素，包括从像最大的相似之处病理像可用性，transfectability和生长特性只是实际问题的临床需求。重要的是，前，继续进行1应该确保融合的3&#39;端非编码区的报道基因对嵌合转录物的水平的预期效果。没有来自3中的荧光素酶信号显著差异的&#39;UTR轴承和对照报道构建体瞬时转染在所选择的细胞将指示该3&#39;端非编码区是未官能此细胞模型中，提示输入搜索替代的。对于高通量格式中，3&#39;端非编码区，轴承和控制荧光素酶报道构建体应当稳定地整合在所选择的细胞系。使用稳定整合在瞬时转染细胞系是优选的有几个原因。这将扩大的蜂窝模型，包括难以转染的细胞系的选择，降低在从转染效率的波动所产生的数据的可变性，消退的费用。此外，当瞬时转染的细胞经常超负荷的DNA质粒导致系统的饱和度。在这些设置在记者表达的进一步增加可能是具有挑战性的，导致对潜在的上调化合物的灵敏度下降。 最后，当所有试验组分均成立，它是移动到高通量形式，以确定该测定， 即可行性前关键的，如果荧光素酶活性可以确实上调和下调经由插入3&#39;特异性UTR。最好控制将是报道通过感兴趣的3&#39;非编码区来调节mRNA的稳定性或译小分子。如果有这些是不可能的，替代的控制模仿所希望的效果被接受。这些可以是miRNA的或已知通过结合感兴趣的3&#39;端非编码区，以发挥它们的作用的RNA结合蛋白。评价这种控制前的初级筛选不仅表示显影测定法的功能，而且还允许估计在高通量形式的动态范围，灵敏度和性能。 使用所描述的协议，我们筛选了2000化合物库6。神经母细胞瘤，婴儿期最常见的颅外实体肿瘤，被用作生物模型和MYCN癌基因，其扩增的3&#39;端非编码区强烈预示神经母细胞瘤的不良结果，作为目标基因7。 图2列出了实验布局。筛选被分为三个运行与27板筛选一次运行该批处理大小。本批27片包括光谱采集库板N.1-N.8 + n.25（第一轮），N.9，n.16 + n.25（第二轮），并n.16，n.24 + n.25（第三轮），每个板筛选一式三份。因此，一个库板（n.25）所有三种运行使得可能相互比较的运行范围内测定。以下协议描述了一个单，一式三份测试9库板运行。

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			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:239px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:263px;">Culture plate 96-well White</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6005688</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">StorPlate 96-well U bottom (dilution plate)</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6008190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">100 ml disposable trough for reagents</td>
			<td style="width:239px;">Tecan</td>
			<td>10 613 048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">300 ml disposable robotic reservoir</td>
			<td style="width:239px;">VWR</td>
			<td style="width:119px;">PB12001301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 50&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 200&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td align="right">3711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ONE-Glo Luciferase Assay System&#160;</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">E6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">RPMI</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE12-918F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">PBS-1X, w/o Ca++, Mg++</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">L-Glutamine 200mM</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">The Spectrum collection (compound library)</td>
			<td style="width:239px;">MicroSource Discovery Systems</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan Freedom EVO200 robot&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan F200 multiplate reader&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

转录和转录后调控对基因表达产生深远的影响。然而，普遍采用的基于细胞的筛选试验集中于转录调控，而实质上旨在启动子靶向分子的鉴定。其结果是，转录后机制在很大程度上未覆盖由基因表达的靶向药物的开发。在这里，我们描述了，并在确定能够修改它的化合物旨在调查的3&#39;非翻译区（3&#39;非编码区），在其mRNA的命运的调制的作用的基于细胞的测定。该测定法是基于使用含有感兴趣的基因稳定地整合入一个疾病相关的细胞系的3&#39;非编码区一个萤光素酶报道构建体。该协议被分成两部分，与最初的焦点上的初级筛选旨在治疗24小时后影响萤光素酶活性的分子的鉴定。该协议的第二部分介绍了反筛选必要区分化合物特别是通过3&#39;UTR调控荧光素酶的活性。除了详细协议和有代表性的结果，我们提供关于该测定的开发和上内源靶的命中（多个）的验证重要的考虑因素。所描述的基于细胞的报告基因分析将帮助科学家识别分子通过依赖于3&#39;UTR转录后调节机制蛋白质水平。

使用所描述的方法，我们筛选了2000-化合物库的转录后控制机制的潜在调节剂通过MYCN基因的3&#39;非编码区施加。 图3示出了初级筛选由单个库板例举的结果。显示为媒介物处理的对照的百分比荧光素酶信号，通过测量荧光素酶活性与单个库板的化合物处理CHP134-mycn3UTR细胞的一式三份板获得。正如预期的那样，多数化合物对荧光素酶活性没有影响由接近100％基线值所示。命中（明...

Watch this Scientific Journal Video about High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes at JoVE.com

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

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 ...

high-throughput-screening-for-chemical-modulators-post

Research

JoVE Journal

Biology

9.1K 次观看.  University of Trento. 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’ UTR.

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

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 ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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 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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.