This research was supported by Advanced Research Projects Agency - Energy (ARPA-E) Award No. DE-AR0000313.

1.悬浮培养的建立<ol><li> BY-2介质通过加入4.43克Linsmaier＆Skoog基础介质30克蔗糖，200毫克KH 2 PO 4制备的液体，和200微克至900的2,4-二氯苯氧乙酸（2,4-D）毫升蒸馏水水和pH至5.8，用0.1M的KOH。调节pH值之后，调节最终体积至1000毫升蒸馏水和高压釜中。介质可以在4℃保存最多2周。 </li><li>接种用100ml液体BY-2的媒体和单片BY-2愈伤组织（&gt; 1厘米直径）上生长固BY-2介质的250ml Erlenmeyer烧瓶（液体BY-2的媒体与添加1％琼脂）中，用铝箔密封。在28-30℃下孵育培养振摇5天。 注:BY-2愈伤组织被保持在为长期贮存的固体介质，如液体培养物将迅速长满。培养体积可以根据需要进行调整，通常的100毫升培养将能够装载三十3个6-WELL板。 </li><li>转让2 ml对数相BY-2培养到98毫升的新鲜BY-2培养基中，并在28-30℃振荡孵育5-7天。 注意:100稀释的5-7天龄培养物，而不是与愈伤接种:建立液体培养物的传代培养可以定期使用1来进行。 </li><li>调匀的培养，因为细胞将迅速沉降，并转移6毫升细胞培养物至15ml锥形底离心管，并让细胞沉降至少10分钟。通过除去上清液调节红细胞体积与总体积的50％。 </li><li>通过反相和吸管500微升成用于消化一个6孔板的每个孔中摇管中。使用宽孔移液管在此阶段转移的细胞，因为它们是致密的并且会阻塞标准枪头。 </li></ol> 2.原生质体分离<ol><li>打开所述机器人系统（ 图1A）的所有部件，并打开该板移动器任务调度软洁具。任务调度程序集成了其它设备，以使每件设备之间板的传送微板动。 </li><li>之前的实验中，定义为实验室器皿中的协议用于每个片实验室器皿，在板移动器软件的技术规范（ 例如 ，盘，盖），以及开始和结束的位置，如下: <ol><li>从板块发软件主工具栏中单击设置 ，然后选择管理容器类型命令。 </li><li>如果容器类型在现有容器类型库中列出，然后选择适当的容器中。 </li><li>如果容器类型没有在现有的容器类型库中列出然后单击添加以指定新的容器类型。 </li><li>添加新的容器类型后，插入新容器（高度，宽度，堆放尺寸和夹具偏移量）的尺寸到相应的选项卡，然后单击保存 。<ol><li>如果正确的尺寸不能从制造商，然后准确地用卡尺确定所有尺寸的精确到毫米。在测量容器尺寸误差将导致板动机故障。 </li></ol></li><li>重复步骤2.2.2至2.2.4.1对所有类型的容器，该板块发会遇到。此步骤为所有实验器皿完成后，有必要定义用于每个容器（步骤2.2.6）的开始和结束位置。 注意:对于这个协议的实验室器具包括6孔板中，深孔板，和96孔荧光筛选板。 </li><li>以限定每个容器的开始和结束位置，点击特定的协议的开始/结束标签。首先，选择每个容器的起始位置。接下来，检查在开始和结束位置都为带盖/ Unlidded的框。重复所有的容器。 注意:如果集装箱将被留在另一块的EQU双模接收机设备（而不是板发）结束位置可以留空。 </li></ol></li><li>在这个阶段所有平板手动装载到用于整个工作流程的开始位置。用BY-2细胞加载96孔荧光隔离板成酒店2，6孔板进酒店3，96深孔板将用于转化到板加热器/冷却器巢2，和50毫升的锥形含有酶溶液（见补充文件 ，第1.2.2节）到多模饮水机管。 <ol><li>如果变换将在协议来进行，以10微升每孔的质粒DNA的预装载96深孔板（1微克/微升，A 260/280&gt; 1.8）含有OFP报道构建和孵化在板加热器/冷却器在4℃。每个孔将用于单个转换，从而填充将取决于实验设置井的数量。 </li></ol></li><li>装载的自动完成所有物资和板ð液体处理平台，在指定地点，并定义每个项目中的自动化控制软件（ 图1B）的位置。 <ol><li>装载在MMG200μl的40％的PEG（0.4M甘露醇，100mM的MgCl 2的，4毫MES，pH值5.7）在第1列，将200μl碘化丙啶的预装入的96孔板（PI，1微克/毫升）在第2栏，以及200微升的在第3列（巢2）乙醇，和枪头的巢8的盒子。 </li><li>要定义实验室器皿中的自动化液体处理平台的软件，从菜单中的位置，选择工具 ，然后单击编辑实验室器皿 。 </li><li>选择实验室器皿从下拉菜单类型或定义使用新建医学实验按钮，一个新的实验器具类型。 </li><li>定义选择的主要协议选项卡，然后单击配置选择实验室器皿每件实验室器具的位置。确保放置在自动化液体处理平台上的每一块实验室器皿是前运行协议定义。 </li><li>要触发自动协议，单击配置文件资源管理器窗口，然后选择要运行的协议（ 原生质体分离 ， 细胞计数和PEG介导的转化 ）的名称。 </li><li>单击运行初始化将在方案中使用的所有设备。 </li><li>最后，在工作资源管理器窗口中，单击添加工作单位 ，以验证系统中的所有容器/实验室器材，并开始自动协议。 注:的自动化协议完整的描述包含在补充文件。 </li></ol></li></ol> 3.建立标准曲线用于细胞计数<ol><li>在1毫升体积的1×10 6原生质体/ ml的浓度手动集中原生质体。原生质体离心在1000 XG 10分钟，取出上清液。 </li><li>手动添加900微升70％的乙醇（保持在4℃）的原生质体沉淀并重新悬浮沉淀。孵育10分钟在冰上固定细胞。 </li><li>添加的PI 100微升（1微克/毫升）的原生质体来标记细胞核，并允许检测由板阅读器。 </li><li>负载80微升液体BY-2介质插入96孔荧光筛板起始于第2列的各列和行。 </li><li>在96孔荧光屏蔽板的第1列，加入原生质体的70微升和50微升BY-2介质到每个孔中的柱。 </li><li>放置板的自动化液体处理平台的巢6，并运行一个2倍连续稀释。 <ol><li>要进行自动化液体处理平台上的连续稀释，单击启动连续稀释向导并调整传输量（60微升），混合和体积（2，70微升），初始井数（第1列，行AF），稀释井（列2-11，行AF），以及吸入和排出性能（从井底0.5mm）的。 </li><li>设置参数后，保存d运行的协议。 注:由于所有原生质体用的PI标记（由于细胞的固定），荧光正比于原生质体的浓度。 </li><li>连续稀释完成后，从窝9取下板并插入板读数器，并运行在平板阅读器软件的标准曲线协议。 注意:标准曲线将随后由荧光从PI（激发536纳米/发射620毫微米）各孔与已知原生质体浓度进行比较来生成的。 </li></ol></li><li>一旦酶标仪协议完成后，取出板和收集高压灭菌或在生物安全议定书规定"等去振兴程序的任何转基因细胞。 </li></ol> 4.转型的微观分析<ol><li>与转化的原生质体从机器人系统的酒店1（自动协议3， 补充文件的产品）取下板。 </li><li>转在倒置显微镜，照相机和荧光灯。 </li><li>选择用于起始聚焦的原生质体的10X物镜，打开卤素灯，并关闭快门的荧光灯。 </li><li>压板到微观制度和注重利用明原生质体。 </li><li>着眼于原生质体后，关闭卤素灯泡和打开快门的荧光灯。 </li><li>选择CY3 / TRITC过滤器由转基因原生质体表达的蛋白质pporRFP可视化设置。 </li><li>扫描每个孔，以确定原生质体表达pporRFP荧光标记物的数目。 </li><li>计算转化效率原生质体的表达荧光标记物的原生质体的总数量的总数。 </li><li>收集含有生物安全认可袋高压灭菌或在生物安全议定书规定"等去振兴程序转基因细胞的板块。 </li></ol>

作者宣称，他们没有竞争的经济利益。

上述的协议已被成功验证为原生质体分离，计数，以及使用该BY-2烟草悬浮细胞培养物转化;然而，该协议可以很容易地扩展到任何植物悬浮培养。目前，原生质体分离和改造已经在众多的植物，包括玉米（ 玉米 ）10，红萝卜（ 胡萝卜 ）32，杨树（ 胡杨 ）33，葡萄（ 葡萄 ）34，油棕（ 油棕 ）35实现，生菜（ 莴苣 ）36，芥菜（ 雪里蕻 ）37，水稻（Oryza sativa）38，和柳枝稷（ 柳枝稷 ）39,40。而在培养和消化条件的变化因物种发生，在这项工作中开发的协议的条款，替换培养基或改变挖的条件estion不应该改变的基本协议。调整到由操作者协议的初始化之前被加载所需的消化不同的物种，培养基和酶，从而交换这些部件是微不足道的。作为一个额外的好处，该协议是易于进行筛选多种消化条件与由系统测量的原生质体的产率。这是一个重要的组成部分，作为使用成本低的酶是必要的高通量应用，因此，它以最小化在消化中使用的酶的量是重要的。 类似的文化和消化，修改改造方案将采用该系统从不同的植物分离的原生质体的转换时需要。相当多的工作已经在植物的原生质体16,35上的PEG介导的转化的优化进行的，目的是影响转化效率许多因素。在当前的禾影响转化效率RK一个限制因素是二进制测试质粒的大尺寸，16028 bp的，将其用于模拟一个基因组编辑质粒大小的目的。在BY-2原生质体的PEG介导转化以往的研究，40％-60％改造已经实现;然而，这些研究中使用的小5000 bp的质粒41,42。物种之间的主要修改的PEG介导的转化包括PEG和MgCl 2的，量和DNA的浓度的浓度和反应时间。作为PEG和MgCl 2的溶液引入到上试剂板的系统，与该DNA是预加载到深孔板，这些条件能够容易地在上述的协议进行修改。此外，增加或减少培养时间，与混合速度的变形例沿，可以由机器人平台被精确地控制。变换协议的电流限制是缺乏的筛选使用板读数器来检测荧光报道，由于聚乙二醇介导的转化在BY-2原生质体中的低效率。在其它物种，如拟南芥 ，玉米16，PEG-介导的转化是40-90％的效率。在这样的系统中的转化体可以用在机器人上的板读取器被快速表征。因此，这将是可能的阳性转化，以确定井，并且还量化表达水平。对于应用程序，如启动子的筛选，该功能会增加当前协议的效用，并且将在稍后的系统的迭代加入。 除了的转化效率优化BY-2原生质体中，若干修改可能会增加系统的效率和整体吞吐量。原生质体转化，允许原生质体的自动化以前的研究沉降到井的底部，在加入之前^ h的解决方案43。在当前的协议，原生质体混合在板振荡器，以防止沉降和使用平底孔板保持原生质体悬浮液中。此外，转换过程中，原生质体不能在深孔板沉降，以减少暴露于PEG，其可以是过度长时间暴露的毒性。这样，从深孔板转移到96孔荧光筛板时将原生质体稀释至10倍的W5溶液中，产生每孔1.23×10 3±4.66×10 1原生质体。为了增加转化后转移的原生质体的数目，孵育步骤可以被添加，以使原生质体沉降，除去上清液，然后加入液体BY-2的媒体的一个较小的体积。此外，如果基于平板离心机加入到该系统中，这个步骤可以更迅速地实现。可能添加功能到t另一个修改他系统将是使用一个活/死荧光测定法来确定之前变换所述分离的原生质体的可行性。在当前的协议，荧光素二乙酸酯（FDA）测试了作为活染料;然而，BY-2原生质体的背景荧光遮蔽了FDA的信号。如果使用更稳定的存活率染料，然后原生质活力可以被确定为红（PI）的绿色的比率（钙黄绿素AM 等 ）的荧光，和标准化的类似细胞计数协议。这些修改将进一步增加功能的系统，并且将被包含在将来的协议。 至于目前的协议，有必须遵循以确保协议的成功几个关键步骤。首先，BY-2培养物的年龄/健康至关重要保证活原生质体可以被分离用于随后的步骤。如在步骤1.3所解释的，通过在对数期培养转移到新鲜烧瓶1:50比例的细胞的新鲜培养基，并允许培养中生长5-7天，可行的原生质体的最大数量可被分离。使培养物中生长使用指定的接种物将显著降低原生质体产量，并导致随后的协议失败更长或更短的持续时间。第二个关键步骤是步骤1.5，因为使用的，而不是指定的宽口枪头，标准枪头，将导致提示堵塞，并导致被转移不正确的卷。关于机器人系统的设置中，自动化液体处理平台上的板的布置中，步骤2.4，是非常关键的，具有修饰的设置防止液体处理程序的头到达上的6孔板的所有孔中。由于自动化液体处理平台的设计中，6孔板必须放置在窝5中，或选择用于此仪器的协议将失败。同样，未能正确地在盘定义实验室器具发软件，2.2步，将导致仪器无法抓住，掉落导致协议终止的实验室器具。对于细胞计数协议中，关键的步骤包括用乙醇，步骤3.2细胞的固定。如果细胞不之前用PI标记固定的，只有不能存活的细胞将被标记，代替原生质体的全部人口，并且它将不可能计数分离的原生质体的数目。最后关键步骤，步骤4.6，涉及用于pporRFP荧光标记物的表达的显微镜分析荧光滤波器的选择。使用其他的"红色"过滤器集允许从叶绿素荧光背景，显著增加所有含有叶绿体原生质体信号，防止转化效率的计算。仔细秉承规范在这些关键步骤，保证了成功，最大的机遇是最可重复的数据更代等。

近年来，一直放置在转基因作物的设计克服了各种疾病1，赋予抗除草剂2，赋予干旱3,4和耐盐5显著动力，防止虫食6，提高生物产量7，降低细胞壁顽抗8。这种趋势已经通过新的分子工具的发展，用于产生转基因植物，包括使用CRISPR和TALENS 9基因组编辑，基因通过的dsRNA 10，miRNA的11，和siRNA 12沉默辅助。虽然这些技术简化了转基因植物的一代，他们还创建了一个瓶颈，其中产生的转基因植物的数量之多，无法使用依赖于植株再生的传统系统进行筛选。与此相关的瓶颈，而沉默和基因组编辑构建体能够迅速地插进植物，许多目标性状不能产生所期望的效果，这通常没有发现直到植物在温室中进行分析。在这项工作中，我们开发了用于植物原生质体的快速，自动化，高通量筛选的方法，具体而言，以解决在大量的基因组编辑和基因沉默靶的早期筛查的当前瓶颈。 使用原生质体的，相对于完整的植物细胞，具有几个优点对自动化平台的开发。首先，原生质体的植物细胞壁的消化后分离，以及与此屏障不再存在，转化效率提高13。在完整的植物细胞，只有两种用于转化很好地建立的方法，基因枪法14和农杆菌介导的转化15。这些方法都不能够容易地转化为液体处理平台，生物射弹需要TRANSFO专门的设备细则第十五，而农杆菌介导转化需要共培养和随后的去除细菌。无论是适合于高通量方法。在原生质体的情况下，转换是经常使用聚乙二醇（PEG）介导的转染16，其仅需要几个溶液的交流，并非常适合用于液体处理平台进行。第二，原生质体，顾名思义，是单细胞培养物，从而与在植物细胞培养物结块和链的形成相关的问题，没有在原生质体观察。在使用基于平板分光光度计，细胞凝集快速筛选而言，或细胞在多个平面将导致难以取得一致的测量。因为原生质体也比其培养基稠密，它们沉淀到孔的底部，形成一单层，这有利于对基于板状光度法。最后，尽管植物细胞悬浮培养物是PRIMAR随手从愈伤组织17衍生的原生质体可以从多种植物组织的收获，从而导致识别组织特异性表达的能力。例如，能够分析根级或一个基因的叶特异性表达可以是表型的预测很重要的。由于这些原因，在该工作开发的协议使用从广泛使用的烟草（ 烟草属）"亮黄色&#39;2（BY-2）悬浮培养物分离的原生质体进行了验证。 在BY-2悬浮培养已被描述为高等植物的"的HeLa"细胞，因为在植物细胞中18的分子分析其无处不用途。最近，BY-2细胞已被用于研究植物的影响压力19-22，细胞内蛋白质定位23,24，以及基本的细胞生物学25-27表明在植物生物学这些培养物的广泛的实用性。 BY-2培养物的一个附加的优点是到培养物与阿非迪霉素同步，这可导致增强的基因表达的重现能力研究28。此外，已开发了用于使用低成本酶29,30 BY-2原生质体的提取，如传统上用于产生原生质体的酶是成本过高的高通量系统。因此，下面描述的协议已被使用BY-2悬浮培养验证，但它应该是易于进行的任何植物细胞悬浮培养。证明的概念实验使用CaMV 35S启动子的控制下，从硬盘珊瑚滨橙色荧光蛋白（OFP）报告基因（pporRFP） 滨 31进行。

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Orbitor RS Microplate mover</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:185px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Bravo Liquid Handler</td>
			<td style="width:157px;">Agilent</td>
			<td style="width:185px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Synergy H1 Multi-mode Reader</td>
			<td style="width:157px;">BioTek</td>
			<td style="width:185px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">MultiFlo FX Multi-mode Dispenser</td>
			<td style="width:157px;">BioTek</td>
			<td style="width:185px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Teleshake</td>
			<td style="width:157px;">Inheco</td>
			<td>3800048</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">CPAC Ultraflat Heater/cooler</td>
			<td style="width:157px;">Inheco</td>
			<td>7000190</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:255px;">Vworks Automation Software</td>
			<td style="width:157px;">Agilent</td>
			<td></td>
			<td style="width:167px;">Software used to control and write protocols for Agilent Bravo</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:255px;">Momentum Software</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td></td>
			<td style="width:167px;">Task scheduling software for controlling Orbiter RS</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:255px;">Liquid Handling Control 2.17 Software</td>
			<td style="width:157px;">Biotek</td>
			<td></td>
			<td style="width:167px;">Software used to control and write protocols for MultiFlo FX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">IX81 Inverted Microscope</td>
			<td style="width:157px;">Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Zyla 3-Tap microscope camera</td>
			<td style="width:157px;">Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">ET-CY3/TRITC Filter Set</td>
			<td style="width:157px;">Chroma Technology Corp</td>
			<td>49004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Rohament CL</td>
			<td style="width:157px;">AB Enzymes</td>
			<td style="width:185px;">sample bottle</td>
			<td>low-cost cellulase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Rohapect UF</td>
			<td style="width:157px;">AB Enzymes</td>
			<td style="width:185px;">sample bottle</td>
			<td>low-cost pectinase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Rohapect 10L</td>
			<td style="width:157px;">AB Enzymes</td>
			<td style="width:185px;">sample bottle</td>
			<td>low-cost pectinase/arabinase</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Linsmaier &#38; Skoog Basal Medium</td>
			<td style="width:157px;">Phytotechnology Laboratories</td>
			<td style="width:185px;">L689</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2,4 dichlorophenoxyacetic acid</td>
			<td style="width:157px;">Phytotechnology Laboratories</td>
			<td style="width:185px;">D295</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">propidium iodide</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">P4170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Poly (ethylene glycol) 4000</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">95904-250G-F</td>
			<td>Formerly Fluka PEG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Propidium Iodide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">25535-16-4</td>
			<td>Acros Organics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">CaCl2</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">C7902-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Sodium Acetate</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">BP333-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Mannitol</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">M1902-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Sucrose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">S5-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">KH2PO4</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">AC424205000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">KOH</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">P1767</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Gelzan CM</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">G1910-250G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">6-well plate</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:185px;">103184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">96-well 1.2 ml deep well plate</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:185px;">AB-0564</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">96 well optical bottom plate</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:185px;">165305</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Finntip 1000 Wide bore Pipet tips</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:185px;">9405 163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">NaCl</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">KCl</td>
			<td style="width:157px;">Sigma Aldrich</td>
			<td style="width:185px;">P4504-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">MES</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">AC17259-5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">MgCl2</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:185px;">M33-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a robotic platform for high-throughput protoplast isolation and transformation

在过去的十年里，已经在使用植物原生质体，范围从模型物种作物物种，信号传导途径，转录调控网络，基因表达，基因组编辑，和基因沉默的分析的回潮。此外，显著进展已在植物的原生质体的再生，这已产生在使用这些系统用于植物基因组的甚至更多的利益作出。在这项工作中，一个协议已使用机器人平台已经开发了用于原生质体分离和转化的自动化从&#39;亮黄色&#39;2（BY-2）烟草悬浮培养。使用花椰菜花叶病毒35S启动子（35S）的控制下，为橙色荧光蛋白（OFP）报告基因（pporRFP）的转化方法进行了验证。原生质体OFP表达，荧光显微镜证实。分析还包括使用propidiu原生质体生产效率的方法米碘。最后，被用于原生质体分离过程的低成本食品级酶，绕过用于实验室级的酶，它们成本昂贵的自动化原生质体分离和分析的高通量的需求。根据此工作开发的协议，从原生质体分离到转化完成过程可以在4小时进行中，在不脱离操作者的任何输入。而在这一工作开发的协议是与BY-2细胞培养物中证实，程序和方法应该是可平移的任何植物悬浮培养/原生质体系统，该系统应使作物基因组学研究的加速度。

在目前的研究中，BY-2的倍增速率从14-18小时取决于在该培养物温育的温度，用15小时的平均细胞周期长度的先前的报告一致变化。与此倍增速率，以1:100起始接种物用于启动培养，导致培养物在5-7天的50％的收集细胞体积（PCV）。在当前的协议，其中培养物在200ml培养基中生长，在7天，这提供了足够的细胞以填充33 6孔板生成100ml的PCV。在原生质体产率而言，产生在协议中规定的条件消化4.70×10 5±5.77×10 3个原生质体每孔，导致在2.82×10 6个原生质体每6孔板（ 图2A）。根据这些数据，214 96孔板（70微升每孔的原生质体）可以由单一的6孔板消化被加载，以fo的电位被同时消化v提供两种6孔板中，利用两个板状加热器/冷却器电台。因此，可以在单个三小时消化协议过程中产生的原生质体的最大数量是5.64×10 6。 而每消化协议生成原生质体的总数为系统提供一个度量，它也是必要的，因为原生质体通过协议的不同步骤转移到评估在原生质体的浓度的损失。这样，原生质体的浓度在协议的每个转印步骤之后进行评价。消化协议之后，1.15×10 4±1.35×10 2的原生质体转移至各孔的96深孔板中，如果在进行转化方案，或96孔荧光筛板（ 图2B）。一旦改造方案已经完成，1.23×10 3±4.66点¯x10 1的原生质体转移到96孔荧光筛板（ 图2C）。除了转移的原生质体的数量，重要的是要确定该原生质体的可行性，以确保多个移液阶段没有损坏原生质体。 基于从活力测定结果，原生质体的95.1±0.04％的消化后存活，转移到96孔荧光屏蔽板后92.1±0.06％，和转换协议后91.7±16.67％。尽管多步移液操作，没有任何样本之间没有显著差异（P&gt; 0.44）。除了这一措施生存能力，协议被用于通过测量使用PI染色原生质体的总数确定在该过程的任何阶段原生质体的浓度开发的。正如图3所示，标准曲线是发电机密封通过测量荧光强度为从系统的原生质体的已知浓度编标准曲线是一个很好的配合（R 2 = 0.967）与原生质体从每孔0〜30000的原生质体跨越范围的总数。为了验证标准曲线，用已知的数原生质体（8,125和24,375使用血球计）的样品的荧光强度，得到与从标准曲线的方程计算原生质体的数目。基于这些结果，绘制标准曲线预测在每9,560和28200原生质阱，15和14％的误差。考虑到还存在使用血球测量细胞数变异，这种误差被认为是可以接受的。 除了细胞数和存活力的结果，变换协议成功地产生转基因细胞中表达OFP（ 图2D - ˚F）。不幸的是使用的协议导致低，〜2％，转化效率，由于在此研究中，并且由于协议没有为BY-2原生质体中特别优化所使用的大16028 bp的二元质粒。为BY-2原生质体的转化方案，在试剂和条件具体而言的优化，预期增加转化效率，如在讨论阐明。在这个协议条款，在转化过程旨在演示从原生质体分离转型证明了概念道路，并成功地实现这一目标。 成功验证所有单个程序后，从原生质体分离转型协议的时间过程，获得指标。如在图4表明，时间的重大投资在消化阶段，在其中需要FO 3小时花费r是原生质体的细胞壁和释放的完全消化。在此过程中，18个循环是在板振荡器和板式加热器/冷却器1孵育站之间运行，与板动子移动在两个站之间的板。在板振荡器和板式加热器之间的传输时间/冷却器1只需要8.9秒，并确保大部分消化过程都花在孵育和晃动。从过程的开始时的总时间由多模式分配器来完成酶的负载量为2.2分钟。总体而言，完整的原生质体分离协议在3小时和22.8分钟内完成。基于这些结果，在原生质体分离协议的88％用于消化。的原生质体分离协议完成后，转换协议启动，和27.5分钟内完成。类似于原生质体分离协议，在转化过程的72％用于孵育反应。在协议的唯一一步具有显著时间组分（&gt;的总过程时间的10％）是由自动化液体处理平台，占的过程中的总时间的14％的协议的初始化。在这种方式中，协议的86％用于在初始化和孵化，只有14％的用在吸液和转移步骤的时间。原生质体分离和转化的完整持续时间为3小时，50分钟和53秒，从操作者无需外部输入。 <img alt="图1" src="/files/ftp_upload/54300/54300fig1.jpg" /> 图 1: 示意图机器人平台和自动化液体处理平台的配置协议中的 （A）的机器人系统组成，可以通过印版移动设备访问3板垛/酒店。酒店1是指定的封盖/解封装站，酒店2是96孔荧光板叠，酒店和3是6孔板栈。该系统具有两个液体处理器，一个自动化液体处理平台，以及喷嘴的基于多模分配器。对于加热/冷却该机器人平台具有两个板的加热/冷却单元，用板振荡器混合所述板沿。最后，该系统具有用于测量荧光的基于单色酶标仪。所有组件都装在一个定制的生物安全柜。 （B）调用一个试验性协议之前启动自动化液体处理平台与试剂板和提示框占据巢2放置在自动化液体处理平台鸟巢8（C）配置的配置。深孔板位于窝4，96孔荧光筛板位于窝6，含有原生质体6孔板位于鸟巢5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54300/54300fig1large.jpg" target="_blank">请点击此处到v</a><html> IEW这个数字的放大版本。 <img alt="图2" src="/files/ftp_upload/54300/54300fig2.jpg" /> 图2: 每个转印之后，用橙色荧光蛋白报告基因转化后BY-2原生质体的代表性图像 （A - C）的显微照片BY-2原生质体密度在6孔板，70转印后的96孔板。微升从6孔板，和96孔荧光筛板变换后。 （D）明改造后BY-2原生质体的显微照片。 （E）荧光BY-2原生质体表达OFP基因使用的Cy3 / TRITC过滤器设置可视化。 （F）明的覆盖和荧光显微照片展示转化效率低。比例尺= 50微米。<html>目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54300/54300fig3.jpg" /> 图 3: 用于计算原生质体的数目的标准曲线在给定井的原生质体的数目可以通过比较荧光强度，以任意单位（AU），该方程的标准曲线来计算。原生质体数目的验证是在图形上的指示，从板读数器确定9561原生质体，和8,125算的上血球点标准曲线和血球进行比较。总体而言，15％的误差，发现两种技术之间。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54300/54300fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 重4"SRC ="/文件/ ftp_upload / 54300 / 54300fig4.jpg"/&gt; 图 4: 甘特图用于生产BY-2原生质体和遗传改造的所述步骤的大部分协议，〜3小时，被用在BY-2细胞的消化来产生原生质体。酶溶液和失活的装载仅需要12％的总过程时间。类似地，在板振荡器转化反应的温育占变换协议的72％。总体而言，原生质体分离，直到遗传转化实现的过程是在4小时（3小时，50分钟和53秒）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54300/54300fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充文件:用于原生质体分离，细胞计数和PEG介导的转化自动化协议&lt;。/ STRONG&gt;的机器人平台来完成上列每个都包含在这个文件中的任务而开发的完整协议。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54300/Supplemental_File_Revised.docx" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about A Robotic Platform for High-throughput Protoplast Isolation and Transformation at JoVE.com

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

高通量，自动化，烟草原生质体的生产和改造的方法进行描述。该机器人系统可以在模型BY-2系统应该是翻译到非模式作物大规模并行基因表达和发现。

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

a-robotic-platform-for-high-throughput-protoplast-isolation

Nanyang Technological University

Research

JoVE Journal

Genetics

14.2K 次观看.  University of Tennessee, Knoxville. 高通量，自动化，烟草原生质体的生产和改造的方法进行描述。该机器人系统可以在模型BY-2系统应该是翻译到非模式作物大规模并行基因表达和发现。 

视频: A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...