RSM从美国​​国家科学基金会研究生研究奖学金承认资金。 MBN承认从路易斯 - 西格莱尔奖学金资助和彼得·刘易斯赋予的礼物。 DB确认来自美国国立卫生研究院（GM046406）普通医学科学中心的定量生物学（GM071508）国家研究所的资助。我们承认克里斯蒂娜DeCoste与流式细胞仪的实验援助。

图2提供的协议部分1-3的示意图。 1。创建申请进度查询设施的<ol><li>创建引物进行PCR扩增目的DNA结合结构域。同源性ACT1的启动子和激素受体 ​​（如在图3和图4描述的）是通过PCR加入到促进正确重组到应变yMN15。 </li><li>使用高保真聚合酶的PCR扩增感兴趣的DBD。 </li><li>变换&gt; 1微克纯化的PCR产物进酵母采用醋酸锂法13。 </li><li>下面的转换，旋转细胞以最快的速度为15秒，离心，取出改造组合。 </li><li>重悬在约200微升无菌水和板在YPD。 </li><li>第二天，从YPD复制品板放置5-FOA平板（5-FOA板作为冷泉港酵母手册中所述制成）13。 </li><li>允许增长2-4ÐAYS，并重新划至少5-10集落至YPD。使用表1和序列的引物，以验证列入一个菌落PCR反应。 </li></ol> 2。创建ATF质粒记者<ol><li>确定ATF（最有可能来自于文献）的结合位点。 </li><li>为了期望的结合区作为寡核苷酸（或Ultramers，如果需要的话）。顺序的顶部和底部股与NotI位和XbaI位正确出挑如表2所示。 </li><li>稀寡核苷酸对等摩尔浓度（100μM）。 </li><li>磷酸化，使用T4多聚核苷酸激酶，然后退火的热循环仪。反应条件示于表3。 </li><li>月刊〜1-2 pMN8微克用NotI-HF与XbaI位为1小时，在37℃（反应条件在表4）。 </li><li>凝胶净化骨干带。 </li><li>稀释纯化的骨干，10纳克/微升。 </li><li>稀双链，磷ATED结合位点，以每微升5倍骨架的摩尔浓度。 </li><li>用T4 DNA连接酶，结扎结合位点进入消化骨干在室温下30分钟（ 表5）。 </li><li>对于转型，加入半连接反应，50μL的标准，化学感受大肠杆菌如XL1-蓝或DH5-α。 </li><li>热休克细胞45秒在42℃水浴中，然后置于冰上2分钟。 </li><li>加入900微升SOC培养基到转化反应。 </li><li>生长于37℃下进行1小时的辊筒。 </li><li>传播每磅+ AMP（100微克/毫升）板100-200微升细胞孵育过夜。 </li><li>长大E.大肠杆菌在LB + AMP的液体和收获的质粒DNA。序列使用该引物5&#39;-GTACCTTATATTGAATTTTC-3&#39;连接的菌落验证新的报告质粒。 </li><li>变换新的报告质粒到含有ATF-STR酵母AIN使用醋酸锂转化第一节。 </li></ol> 3。量化ATF感应的影响基因表达的利用GFP记者制备样品： <ol><li>让25毫米β-雌二醇股票（10毫升乙醇+0.0681克β-雌二醇）。冷藏保存。 </li><li>成长ATF +记者 - 包括应变过夜5毫升SC-URA。 </li><li>获得9250毫升摇瓶和25 mL的SC-URA到每个。 </li><li>添加0纳米，0.1纳米，1纳米，10纳米，100纳米，1微米或10微米β-雌二醇。这是最容易通过将25mM的β-雌二醇股票的10倍系列稀释液进行。为10μM的β-雌二醇的终浓度，加入10微升25mM的β-雌二醇库存至25 SC-URA中的溶液。 </li><li>加入125μL过夜培养，以培养瓶（1:200稀释）。 </li><li>对于剩下的2瓶，接种与本构关系的GFP生产TG1的n和应变缺乏绿色荧光蛋白。这些都需要用于校准流式细胞仪。 </li><li>摇在200转，在30℃12-18小时。 注：最佳孵育时间由GFP感应不再变化如下补充β-雌二醇的标识时确定。 </li><li>取500微升每一种文化的和独立的1.7毫升离心管降速。 </li><li>吸SC-URA。 </li><li>在1ml的PBS 0.1％吐温20和吸液洗一次。 </li><li>加入1 ml的PBS +0.1％Tween-20的重悬的。 </li><li>添加1ml的PBS 0.1％吐温20的Falcon管中。 </li><li>加入重悬细胞至5ml管（最终体积= 2毫升）（可以储存在4℃下进行数天）。 </li><li>可选：超声细胞轻言分手成群的任何细胞。 流式细胞仪分析：概述：10,000-100,000细胞的荧光每个样品通常分析。数据可以在FlowJo或者使用MATLAB中的自定义脚本或处理R.确保使用GFP阳性和阴性对照来校正GFP通道的电压。一旦FCS文件导出，脚本，如1在图6中沿与功能fca_readfcs（ <a href="http://www.mathworks.com/matlabcentral/fileexchange/9608-fcs-data-reader">http://www.mathworks.com/matlabcentral/fileexchange/9608-fcs-data-reader</a> ）可以被用于处理数据。 </li><li>进行流式细胞仪。 <ol><li>使用前向散射（FSC）和侧向角散射（S​​SC），以确定酵母细胞。 </li><li>流速设置为〜3,000个细胞/秒。 </li><li>测量绿色荧光蛋白（FITC通道）。 </li><li>一旦数据被收集，导出。FCS文件</li></ol></li><li>加载MATLAB。 </li><li>移动，FCS文件，fcs_readfcs.m和类似于从图6到同一文件夹的脚本。 </li><li>改变当前工作目录到包含数据和脚本的文件夹。 </li><li>从图6运行脚本（注意：传说已手动输入，以产生什么如图7所示）。 </li></ol> 4。量化ATF诱导对细胞生长的影响<ol><li>从冻结的股票，连胜了两个（1）含ATF应变和（2）的ATF-缺乏应变（如yMN15）到单独的YPD平板。 </li><li>后第2天生长，接种含有与每个应变5毫升YPD液体的分离培养管中并在30℃生长过夜</li><li>进行10倍的在100％乙醇中的0.25毫β-雌二醇原液高达10,000倍（0.025μMβ-雌二醇）系列稀释。 </li><li>加入40μL过夜培养至10 ml YPD液体（1-250稀释）的。 </li><li>对于每种菌株，加96微升稀释的细胞，以18孔的96孔板的。 </li><li>加入4微升β-雌二醇的细胞中。一个重要的一点是确保每个孔具有总乙醇的量相同。 （备选地，莫<html>重的β-雌二醇浓度的储备可用于随后稀释至点乙醇对生长的影响是不能测定）。 </li><li>执行一式三份。三个孔中的每个应变的应用乙醇 - 仅控制。 </li><li>覆盖96孔板中的气体可渗透膜。 </li><li>监测生长（OD 600）在酶标仪。 </li><li>量化使用任何数目的标准方法，例如寻找最大斜率的增长率。 注：我们已经与开源软件包grofit 14，它运行在R编程环境良好的成功值得一提的是，虽然从96孔板检测计算的增长率相比，摇瓶（25毫升培养）。不一定是相同的（由于在生理和仪表都不同），我们观察到，相对生长速率是相似的（尽管这可能不总是这种情况）。 </li></ol>TLE“&gt; 5。建立诱导基因组等位基因<ol><li>获得pMN10，一个noncentromeric质粒与从pMN8的ATF-反应性启动子融合到KanMX（在相反的方向）。 </li><li>限制性消化载体骨架和凝胶提取物，如第2。 </li><li>退火，磷酸化，并在第2节所述含有适当的结合位点结扎寡核苷酸。 </li><li>序列验证。该质粒将现在作为创建的诱导型等位基因的DNA模板。 </li><li>获得表达目的对应的ATF的酵母菌株。 </li><li> PCR扩增用引物由表6的KanMX -启动子盒（〜2 KB）。 </li><li>变换的PCR产物进用醋酸锂法ATF的表达株。 </li><li>板在YPD和副本板到YPD + G418的第二天。 </li><li>确认使用正向引物（5&#39;-CTGCAGCGAGGAGCCGTAAT-3&#39;）和反向引物的内部对TH启动子的适当的插入E基因的启动子已被替换。反向引物通常设计为200-300 bp的第一个ATG 15的下游侧之间。最终的PCR产物为〜1.2kb的。 </li></ol>

<ol>
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McIsaac，诺伊斯和特司特因（与他人）已提交了该系统的一部分作为专利公开。

此处所描述的基于激素的开关具有无数应用，从研究天然细胞生物学测试感兴趣的DNA结合结构域的目标DNA序列在体内的能力。该系统具有许多理想的特性，包括分级响应诱导，快速的行动，并严格监管（ 即 ，没有可测量的泄漏，参见图7）。然而，仍然有改进的空间和扩展。 最近在合成生物学的工作强调多路 ​​合成系统和扩大良好的特点，可编程的DNA部分16的剧目。鲜明的靶基因的独立，条件表达式既需要多个诱导和缺乏特异性重叠的DNA结合结构域。工程和天然受体的可用性提供了大量部件，用以开发新的人工转录因子。扩大相互正交的开关剧目已被定向进化极大的帮助接近17,18。目前正在努力重新设定我们的人工转录因子的配体结合的特异性将呈现的未来。 先前，基因缺失/过表达的表型影响已经被广泛地研究了在酵母19,20。然而，它并没有被直接地研究表达对不同表型的范围内表达水平的影响。本文介绍的系统的一个主要特点是它的分级性质（ 图8）。虽然通常认为，基因表达和利益表型之间的关系不一定是单调的。人工转录因子例如Z 3 EV和Z 4 EV会使测定的表型反应基因表达的变化简单的任务。 最后，讨论协议编辑在该原稿是用于工程和表征该响应β-雌二醇人工转录因子，然而，一个重要的替代化学反应域是使用可见光响应型结构域（如PhyB/Pif3，LOV，并BLUF）其活动是可逆的，而无需扰乱培养生长培养基21-23。光为基础的系统便于时空控制基因表达，蛋白-蛋白相互作用，离子运输，和酶活性，已证明这是有力21-26。 总之，我们已经提出了诱导，人工转录因子的快速建设在酵母的方法。这个协议提供了其与同源GFP记者结合结构，以及用于使用流式细胞仪分析该报道的内容并测定生长ATF活化的影响的方法的模板。

在酵母中发展基因开关是在学术和工业研究的极大兴趣。在理想情况下，遗传开关可用于仅当由实验者期望激活特定基因（或一组基因的）。 A基因的编码序列的合成启动子的下游放置不表达在不存在诱导分子：在诱导剂除了该基因应迅速表达。它只是在最近已经证明，这样的开关可以在酵母中，在那里在没有诱导物，该菌株显示缺失的表型，但在诱导剂的存在下，表达被成比例地诱导物的水平在所有细胞中激活被改造1,2。从历史上看，在酵母中的条件表达式系统已在营养响应的DNA序列，包括MET，河粉 ，和GAL启动子（其活动的蛋氨酸，磷酸盐外水平敏感，严重依赖半乳糖，分别）。虽然条件表达式可以实现的，它是以牺牲显著多效的成本​​。 激素受体 ​​，如人雌激素受体 ​​已被证明是在真核生物3,4有效开关。在无激素的，稠合到一个激素受体的蛋白可以被隔离在那里它与热休克蛋白90分子伴侣复合物相互作用的细胞质中。在结合适当的配体，受体发生构象变化，导致它从热休克蛋白90分子伴侣复合物释放并露出了核定位信号。观察到的特定激素含有受体蛋白的定位动力学，我们创建Gal4dbd.ER.VP16（GEV，含有Gal4p DNA结合结构域，人雌激素受体的配体结合结构域的合成转录因子的C-末端融合和VP16）与绿色荧光蛋白2。核定位跟随加在8分钟内观察饱和的诱导剂，β-雌二醇的量，到野生型酵母完全是惰性的。到了这个时候，大部分细胞具有极值分布的靶基因（通过荧光原位杂交检测）清晰可见活跃的转录位点，以及完全成熟的mRNA 2,5。 GEV靶基因表达&gt; 2倍，2.5分2,6（使用微阵列测量）范围内增加，这表明它需要&lt;2.5分钟的嵌合体活化剂转运到细胞核，与DNA结合，并激活转录。 而其他几个上开关已在酵母中，利用各种小分子或药物（包括大肠埃希氏经典的多西环素为基础的开关大肠杆菌 7,8和拟南芥靛蓝为主开关9），没有已经实现了应用的速度，特异性，或调节松紧度的激素为基础的开关展出。值得一提的吨帽子快速关断开关也已开发在酵母中，并且通常通过融合基因对蛋白酶或泛素连接酶靶向序列2,10,11,12工作。迅速从细胞中移除一个目标蛋白质的能力，有利于必需基因的研究和基因时，删除10是易受遗传抑制。 在此协议中描述的方法是建立和开关在酵母中合成表征。首先，我们将展示如何快速生成基因组整合的融合蛋白组成的利益的DNA结合结构域，配体结合人类雌激素受体的结构域，与VP16（DBD-EV的）。按照我们的协议，该融合蛋白是从ACT1启动子表达。然后，我们展示了如何创建一个同源报告质粒的ATF和使用流式细胞仪测试其功能。虽然我们设想这些报告质粒被用新的申请进度查询设施（ 图1）使用时，它们可以bË作为记者的原生转录激活为好。最后，提供了详细的试验在96孔板ATF活化对细胞生长的影响以及工程可诱导基因的等位基因。

<table border="0" cellpadding="0" cellspacing="0" width="352">
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		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Name of the Reagent</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:88px;">Comments&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:88px;">Expand High Fidelity PCR System</td>
			<td style="width:88px;">Roche</td>
			<td style="width:88px;">11 732 650 001</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">T4 DNA Ligase</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">M0202S</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Competent E. coli</td>
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			<td style="width:88px;">18258-012</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Estradiol</td>
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			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Ampicillin</td>
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			<td style="width:88px;"></td>
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		<tr height="51">
			<td height="51" style="height:51px;width:88px;">T4 Polynucleotide Kinase</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">M0201</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">PBS</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">10010-23</td>
			<td style="width:88px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:88px;">Tween-20</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">9005-64-5</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">clonNAT</td>
			<td style="width:88px;">Jena Bioscience</td>
			<td style="width:88px;">AB-102L</td>
			<td style="width:88px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:88px;">XbaI</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">R0145S</td>
			<td style="width:88px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:88px;">NotI-HF</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">R3189S</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">CutSmart Buffer</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">B7204S</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Glucose</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">D15-500</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Bacto-Peptone</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">211677</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Yeast Extract</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">210929</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Agarose</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">214010</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">100% Ethanol</td>
			<td style="width:88px;">Decon Laboratories</td>
			<td style="width:88px;">04-355-222</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:88px;">G418</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">11811-031</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:88px;">QIAGEN</td>
			<td style="width:88px;">27104</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:88px;">Lithium acetate dihydrate</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">L-6883</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Salmon sperm DNA</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">93283</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">PEG 3350</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">P-3640</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">Plasmids pMN8 and pMN10; yeast strain yMN15</td>
			<td style="width:88px;">Noyes or Botstein labs</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Luria Broth</td>
			<td>Sigma-Aldrich</td>
			<td>L3397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
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			<td height="22" style="height:22px;"></td>
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			<td></td>
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		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Material Name</td>
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			<td style="width:88px;">Comments (optional)</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:88px;">LSRII Flow Cyometer&#160;</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">Various Models</td>
			<td style="width:88px;"></td>
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		<tr height="103">
			<td height="103" style="height:103px;width:88px;">MATLAB or FlowJo</td>
			<td style="width:88px;">MATLAB or FlowJo</td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Software for analyzing .FCS files from flow cytometry experiments</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">R</td>
			<td style="width:88px;">Open Source</td>
			<td style="width:88px;"></td>
			<td style="width:88px;">For analyzing growth-rate data from plate reader.&#160;</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Falcon Tubes</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">352052</td>
			<td style="width:88px;"></td>
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		<tr height="52">
			<td height="52" style="height:52px;width:88px;">1.7 ml Eppendorf&#160; Tubes</td>
			<td style="width:88px;">GeneMate</td>
			<td style="width:88px;">C3262-1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">250 ml Shake Flasks</td>
			<td style="width:88px;">Corning</td>
			<td style="width:88px;">4446-250</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">96-well, flat-bottom plates</td>
			<td style="width:88px;">Costar</td>
			<td style="width:88px;">3635</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">Plate Reader (Synergy H1 Multi-Mode Plate Reader)</td>
			<td style="width:88px;">BioTek</td>
			<td style="width:88px;">H1MG</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Breathe-Easy Membranes</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">Z380059-1PAK</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Thermocycler</td>
			<td style="width:88px;">various companies</td>
			<td style="width:88px;">Various models</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">SC-Ura powder</td>
			<td style="width:88px;">MP Biomedicals</td>
			<td style="width:88px;">114410622</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">5-FOA</td>
			<td style="width:88px;">Zymo Research</td>
			<td style="width:88px;">F9001-1</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

rapid synthesis and screening of chemically activated transcription factors with gfp-based reporters

合成生物学的目的是合理设计和期望的量化特性构建合成电路，并提供工具来查询本机控制电路的结构。在这两种情况下，对基因的表达进行编程以快速和可调​​谐的方式，没有脱靶效应的能力可能是有用的。我们已经构建了含有URA3盒其次是人雌激素受体 ​​和VP16的配体结合域的ACT1启动子上游的酵母菌株。通过转化该菌株具有线性PCR产物含有对URA3的存在下DNA结合域和选择，组成型表达的人工转录因子（ATF），可以通过同源重组产生。申请进度查询设施工程改造以这种方式可以激活一个独特的靶基因在诱导剂的存在下，从而避免两者的脱靶活性，与常用conditi发现非生理生长条件ONAL基因表达系统。还提供了对绿色荧光蛋白报告质粒，为感兴趣的当地人或人工转录因子的响应特别是快速建设一个简单的方法。

<html> 图5显示了绿色荧光蛋白的诱导作为Z 3 EV，Z 4 EV或GEV随着时间的推移。注意，以响应包含nonyeast DNA结合结构域的申请进度查询设施增加的GFP产量。最近，我们推测，这导致从这些因素中的酵母基因组1实现单基因的特异性。单独GEV感应导致数百个基因的激活/抑制，而Ž3 EV和Z 4 EV含有合适的结合位点（5&#39;-GCGTGGGCG-3&#39;和5&#39;-合成启动子的下游放置仅激活靶基因的表达GCGGCGGAGGAG-3&#39;，分别）1。 图7显示了系统中（）没有诱导结果没有检测到GFP和所有细胞诱导得到响应诱导的Ž3 EV 为诱导GFP通过一系列的能力的严格监管β-雌二醇浓度示于图8。 <html> ENT“FO：保持together.within页=”总是“&gt; <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig1highres.jpg" src="/files/ftp_upload/51153/51153fig1.jpg" /> 图1。人工转录因子与Hsp90的相互作用在缺乏β-雌二醇。的当β-雌二醇结合到ATF，Hsp90的解离，从而使ATF进入细胞核并从质粒记者激活表达。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig2highres.jpg" src="/files/ftp_upload/51153/51153fig2.jpg" /> 图2原理协议包含人工转录因子和建筑/测试同源GFP记者的工程菌株。 load/51153/51153fig3highres.jpg“SRC =”/ files/ftp_upload/51153/51153fig3.jpg“/&gt; 图3中的应变yMN15在LEU2基因座的ACT1pr-URA3-EV序列。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig4highres.jpg" src="/files/ftp_upload/51153/51153fig4.jpg" /> 图4设计的PCR引物，用于扩增目的DNA结合结构域与额外的序列同源性，以产生一个新的融合蛋白时，成功地转化成yMN15。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig5highres.jpg" src="/files/ftp_upload/51153/51153fig5.jpg" /> 通过响应于1μMβ-雌二醇3不同的ATF-启动子对图5。绿色荧光蛋白的诱导 。&lt;/ P&gt; <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig6highres.jpg" src="/files/ftp_upload/51153/51153fig6.jpg" /> 图6。一个MATLAB脚本流式细胞仪的数据处理流程 。这个脚本改编自<a href="http://openwetware.org/wiki/McClean:_Matlab_Code_for_Analyzing_FACS_Data">http://openwetware.org/wiki/McClean:_Matlab_Code_for_Analyzing_FACS_Data</a> 。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig7highres.jpg" src="/files/ftp_upload/51153/51153fig7.jpg" /> 图7。感应响应0μMβ-雌二醇和1微米β-雌二醇以下18小时诱导的GFP用Z 3 EV的。荧光也MEA从细胞缺乏GFP，说明Z轴3 EV系统的严格监管变身。使用代码在图6中生成这个数字。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig8highres.jpg" src="/files/ftp_upload/51153/51153fig8.jpg" /> 图8。诱导剂浓度和表达输出之间的关系与分级〜1的Hill系数（A）分派GFP表达的β-雌二醇浓度的函数。 （二）由（A）分布的平均荧光。该数据是适合的函数G（D）= G 分钟 +（G MAX-G MIN）D N /（K N + D n），其中D是β-雌二醇剂量，G MIN和G是最大和最小最大的GFP值，水库pectively，n为Hill系数，K是能够产生半（G MAX + G MIN）的β-雌二醇剂量。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" width="176"><tbody><tr><td>寡核苷酸序列</td><td>名称</td></tr><tr><td> 5&#39;-TTGAAACCA AACTCGCCTCT-3&#39; </td><td> DBD检查正向</td></tr><tr><td> 5&#39;-tccagagac ttcagggtgct-3&#39; </td><td> DBD选中反转</td></tr></tbody></table> 表1引物用于确认正确融合的感兴趣的雌激素受体 ​​的DBD的。正向引物同源的启动子ACT1和反向引物是同源的雌激素受体 ​​。对于典型的DBD（〜300 bp）的，PCR产物是&lt;1.5 kb的。 <table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr><td>寡核苷酸序列</td><td>名称</td></tr><tr><td> 5&#39;-ggccgc ... DBD暴食网站... T-3&#39; </td><td>热门寡</td></tr><tr><td> 5&#39;-ctaga ...的DNA结合位点反向... GC-3&#39; </td><td>底部寡</td></tr><tr><td> 5&#39;-GCC gcGTGGGCG牛逼GCGTGGGCG G G CGTGGGCGŦGCGTGGGCG摹GCGTG GGCGŦGCGTGGGCG T-3&#39; </td><td>热门寡+ 6X zif268基因结合位点</td></tr><tr><td> 5&#39;-ctagaCGCCCACGCACGCCCACGCC CGCCCACGCACGCCCACGCCCGCC CACGCACGCCCACgc-3&#39; </td><td>底部寡+ 6X zif268基因结合位点</td></tr></tbody></table> 引物用于创建含有双链DNA的利益结合位点见表2出来的，S tructure。要创建Z轴3 EV 为响应启动子，寡核苷酸寡顶+ 6X zif268基因结合位点和底寡+ 6X zif268基因结合使用了。序列建立正确的DNA出挑为小写。该zif268基因的共识结合位点，GCGTGGGCG，带下划线的顶部寡。 <table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr><td>试剂</td><td>量</td></tr><tr><td> T4多核苷酸激酶缓冲液</td><td> 5微升</td></tr><tr><td> T4多核苷酸激酶（@ 10,000单位/ ml） </td><td> 1微升</td></tr><tr><td>顶寡（100μM） </td><td> 1.5微升</td></tr><tr><td>底部寡（100μM） </td><td> 1.5微升</td></tr><tr><td>水</td><td> 41微升</td></tr></tbody></table> 表3磷酸化并在使用50微升上述反应的热循环退火的引物。有两个温度循环器的步骤。第1步：37℃30分钟（磷酸化），和第2步：95℃30秒（下台1℃，每30秒至4℃;退火）。 <table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr><td>试剂</td><td>量</td></tr><tr><td> XbaI位</td><td> 1微升</td></tr><tr><td>的NotI-HF </td><td> 1微升</td></tr><tr><td>质粒DNA </td><td> 5微升（1-2微克） </td></tr><tr><td> 10X切割智能缓冲</td><td> 5微升</td></tr><tr><td>水</td><td> 38微升</td></tr></tbody></table> 表4。质粒pMN8用XbaI和NotI限制性消化。 <table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr height="22" style="height:16.5pt"><td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">试剂</td><td class="xl66" style="width:66pt" width="88">量</td></tr><tr height="43" style="height:32.25pt"><td> T4连接酶BUFFER </td><td> 2微升</td></tr><tr height="22" style="height:16.5pt"><td> T4连接酶</td><td> 0.2微升</td></tr><tr height="43" style="height:32.25pt"><td>骨干@ 10纳克/微升</td><td> 1微升</td></tr><tr height="106" style="height:79.5pt"><td>结合序列@ 5倍摩尔浓度/微升</td><td> 1微升</td></tr><tr height="22" style="height:16.5pt"><td>水</td><td> 15.8微升</td></tr></tbody></table> 表5。结扎结合位点插入质粒DNA。 <table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr><td>底漆</td><td>描述</td></tr><tr><td> 〜40 bp的上游的ATG + CGCACTTAACTTCGCATCTG-3&#39; </td><td>正向引物用于扩增KanMX-启动子</td></tr><tr><td> 5&#39;-反向互补（ATG +〜37bp）+ TATAGTTTTTTCTCCTTGACG-3&#39; </td><td>反向PRIMER放大KanMX启动子</td></tr><tr><td> 5&#39;-caatttgtctgctcaagaaaataaa ttaaatacaaataaaCGCAC TTAACTTCGCATCTG-3&#39; </td><td>正向引物制作GCN4启动子的交换。 </td></tr><tr><td> 5&#39;-tggatttaaagcaaataaacttgg ctgatattcggacatTATAGTT TTTTCTCCTTGACG-3&#39; </td><td>反向引物制作GCN4启动子的交换。 </td></tr></tbody></table> 表6。PCR扩增KanMX，发起人作出titratible等位基因。

Watch this Scientific Journal Video about Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters at JoVE.com

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

本协议描述了实验过程的人工转录因子（申请进度查询设施）与绿色荧光蛋白同源记者和通过流式细胞仪，刺激GFP表达的申请进度查询设施能力量化的快速建设。

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

rapid-synthesis-screening-chemically-activated-transcription-factors

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

14.4K 次观看.  Princeton University. 本协议描述了实验过程的人工转录因子（申请进度查询设施）与绿色荧光蛋白同源记者和通过流式细胞仪，刺激GFP表达的申请进度查询设施能力量化的快速建设。 

视频: Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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 ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

九的原子Deltahedral Zintl离子锗和他们的官能合成有机基团

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.