我们感谢安格伯格实验室协助。这项工作是由马克斯·普朗克学会和耶拿中学的微生物通讯（吉林华微电子）的支持和资助。

1。养虫<ol><li>购买或取得的夜蛾卵离合器北沙参从自己饲养。它们保持在无菌培养皿中，在室温（RT），直到孵化。 </li><li>准备人工饲料饲养如下昆虫: <ol><li>在100毫升开水浸泡500克地白扁豆过夜。 </li><li>添加9.0克抗坏血酸和75g琼脂至1000毫升蒸馏水H 2 O和事后煮沸。 </li><li>让混合物冷却并凝固时（以白色蜡状固体混合物）它在4℃下保存直至使用。 </li></ol></li><li>初孵幼虫转移到一个干净的塑料盒和后部他们在人工饲料在23-25​​℃，长日照政权16小时光照和黑暗8小时以下。每天换食物，直到幼虫经过所有六个龄幼虫。 </li></ol>在代谢活跃的肠道细菌2，13 C-标记的DNA <ol><li>溶解186.1毫克13完全用蒸馏水和去离子水（双蒸2 O）C标记的葡萄糖和100克人工饲料，供对SIP实验拌匀。确保最后的13 C-葡萄糖10毫米浓度的人工饲料。这模仿在S的肠道原位葡萄糖浓度据邵等人在棉花植株北沙参幼虫取食。（提交）。 </li><li>传输9-15健康二龄幼虫的饲养箱含有新鲜的13 C-葡萄糖无菌塑料培养皿（每培养皿1个幼虫）跳涨人工饲料。使用人工饲料含有天然葡萄糖（12 C-葡萄糖）的相同量的，如上所述，用于馈送控制组。 </li><li>发病期间（1天）经常更新的人工饲料。使用饲养条件下，如步骤1.3。 </li><li>每个处理后处理（提取DNA）收集9幼虫。每复制由三个个体构成;使每个处理至少3次重复。 </li></ol> 3。昆虫解剖<ol><li>清洁和消毒的解剖工具，在工作的开始用70％乙醇（工具=维纳斯剪，钳和手术刀细一次性刀片）。 </li><li>取昆虫用软镊子和用蒸馏水洗涤表面上的皮肤。将它们放置在冰上至少30分钟，麻醉它们。 </li><li>虽然麻醉还是他们的地方在0℃，1小时要杀死他们。通过几分钟的浸在乙醇（70％）表面消毒的死虫。 </li><li>执行昆虫解剖到大约15毫升的1X PBS中沉积在无菌培养皿（1XPBS = 137 mM氯化钠，2.7 mM的氯化钾，4.3毫米的Na 2 HPO 4，1.47 mM的KH 2 PO 4的体积，将pH调节至7.4 ，高压灭菌器）。一定有整个虫体完全沉浸在SOLUT的在整个解剖期离子。 </li><li>开始剥离通过使切口在沿右和昆虫的左右两侧的皮肤，开始在头，并完成于肛门。删除整个皮肤，马氏管和脂肪体。切开肠道之前更改为新的缓冲区。部分在需要时在船首，中，后肠和内脏。存储该组织在冷冻（-80至-20℃），直到DNA提取</li></ol> 4。 DNA的提取和扩增<ol><li>将昆虫组织在1.5ml的无菌离心管中，并干燥的所有样品在45℃下在真空浓缩器设备。粉碎干燥组织，用无菌塑料杵。 </li><li>提取使用合适的试剂盒土壤DNA提取的基因组DNA。转干组织试剂盒的反应管，并按照指示由制造商所指示的。 </li><li>确定使用分光光度计最后洗脱的DNA的浓度。 </li><li> VERIF和1492R（5 - GGTTACCTTGTTACGACTT -3） - 通过使用真细菌通用引物27F（AGAGTTTGATCCTGGCTCAG -3 5）制备诊断PCR法雅成功提取昆虫肠道微生物宏基因组的DNA。设置PCR反应如下: <ol><li>设置包含1X缓冲液，1.5毫米氯化镁2，10毫米四种脱氧核苷三磷酸（dNTPs浓度），U Taq酶2.5 DNA聚合酶，各0.5mM引物和60纳克提取的DNA作为模板的50微升反应混合物。 </li><li>使用热循环仪上运行的PCR反应如下:在94℃下进行3分钟初始变性;接着35个循环:94℃45秒，退火55℃30秒，和延伸72℃，1分钟。 10分钟用最后延伸步骤在72℃。 </li><li>验证成功的PCR扩增在1％的琼脂糖电泳的溴化乙锭（EB）染色凝胶（溶解在100毫升的1X TAE缓冲液及污垢加入1μlEB1克琼脂糖）。存款5微升的第ËPCR产物的6倍上样染料进入凝胶袋+加入1μl。 （1×TAE = 40 mM的Tris碱，20mM的冰醋酸，1mM EDTA中，调节pH至8）。 </li><li>使用1xTAE缓冲区300伏115毫安CA运行的凝胶。 20分钟的电泳室。通过使用凝胶文档室（紫外透射连接到一台数码相机和计算机）观察凝胶和您的最终产品的尺寸随还装​​载基因标记进行比较，扩增子的正确大小必须是约1.5 kb的。 </li></ol></li><li>存储PCR产物进行深度冷冻条件下，优先-80℃，直至使用。 </li></ol> 5。分离DNA-氯化铯梯度离心<ol><li>制备的试剂，用于梯度超速离心法，如下所示: <ol><li>通过逐渐溶解603.0克氯化铯在DDH 2 O准备7.163 M氯化铯（CSCL）解决方案，并填补多达500毫升的最终体积。 </li><li>精确解通过对一式三份的三位称量1.0毫升一份termine制备的氯化铯溶液的密度。这通常是1.88和1.89的克/毫升在室温。 </li><li>通过组合50毫升的1M的Tris-HCl（pH 8.0）中，将1ml的0.5M EDTA（pH 8）中和3.75克氯化钾在最终的制备梯度缓冲液（100mM的Tris，100mM的KCl和1mM EDTA）中用双蒸2 O 500ml体积滤器（0.22μm）灭菌和高压灭菌该溶液。 </li></ol></li><li>分离DNA <ol><li>在确定了处理过​​的昆虫的个体的DNA浓度，测定在4.3点，集中对应到复制在一起，并归一化的DNA的浓度，以确保每一个，同样有助于合并样品的那些昆虫。为500-5000毫微克DNA的（再次测量最终浓度）的最终浓度是适合于超离心处理21。 </li><li>要设置梯度介质，结合量化的DNA，梯度缓冲液ð4.8毫升的7.163 M氯化铯溶液一起到6.0毫升无菌的15毫升螺旋盖管（代梯度介质-DNA混合物）的混合量。 </li><li>所制备的梯度介质-DNA混合物小心地放入一个5.1毫升超速离心管（填至管颈的基部）使用注射器和针头。避免抽或形成任何气泡。包括至少一个空白梯度（使用双蒸代替DNA 2 O）中的每个离心机运行作为参考梯度。 </li><li>平衡管对向于每个所需的梯度介质后的10毫克量的填充到各自的超速离心管。密封该管带有"管礼帽"，并确保该密封是无泄漏颠倒试管并施加适当的压力通过手。 </li><li>使用近垂直转子与八井举行5.1毫升超速离心管中离心。仔细密封转子孔并根据毫安加载到转子超速离心机生产商产生的指示。设置超离心条件:旋在50000转（围绕1​​77,000×g离心），温度在20℃，并超速离心运行时间为40小时，用真空。选择最大加速度和减速度不带刹车。 </li><li>一旦完成后，小心地从使用镊子离心机转子取出管子。将它们放置在一个机架中，而不会干扰管道内形成梯度。尽快以尽量减少扩散过程的梯度分离样品。 </li></ol></li><li>通过从分馏等密度梯度分离的DNA提取<ol><li>使用准确的HPLC泵系统来进行梯度分离，这同样分离超速离心管顶部位移法所形成的梯度。 HPLC泵（100％流量梯度）连接到装有1升排量双蒸2 O瓶子，紧紧地附着在打开的泵管与23 G 1的注射器针头。加入足够的bromophenol蓝色染料的双蒸2 O来给它一个深蓝色的颜色。这有利于梯度介质和双蒸2 O的位移之间的界面的可视化</li><li>泵设置为850μL/ min的流速和平衡系统，直至蓝色的双蒸2 O浮现出注射器针头一滴。 </li><li>仔细固定超速离心管到一个夹子支架和刺穿管的底部用注射器23 G 1在长针。该泵连接到超速离心管中插入安装于泵管到管的顶部针，并收集从底部滴直接进入无菌的1.5 ml离心管中。收集的一小部分，每30秒，达每分约425微升，共梯度每12级。请注意，最后分数（分数12）通常包含位移双蒸2 O，应该被丢弃。 </li><li>分馏后，我ASURE使用分析天平中提到的步骤5.1.2所有分离级分的密度。 </li><li>先加入1微升糖原（20微克/微升在DDH 2 O，高压灭菌），作为DNA的低量沉淀的载体沉淀从每个部分（约425微升）的DNA。颠倒混匀很好。 </li><li>新增两卷聚乙二醇（PEG）溶液（溶解150克聚乙二醇6000 46.8 g氯化钠在DDH 2 O在500毫升的总体积（850微升）。过滤，消毒和高压灭菌。的PEG溶液为30 ％PEG 6000和1.6 M氯化钠），并通过反转10次再次混合。 </li><li>离开该管在室温下至少两个小时（如果需要，过夜），以沉淀DNA。 </li><li>离心沉淀物以13,000×g离心30分钟，在RT。一个可见的颗粒应形成。 </li><li>小心地用移液管去除上清并用500μl70％乙醇洗涤沉淀。在相同的离心再10分钟的条件。 </li><li>小心弃去上清液，空气干燥沉淀在室温下15分钟。 </li><li>重新溶解在干燥的DNA沉淀用30μL双蒸2 O，并通过轻轻拍打管拌匀。量化中的每个梯度级分的DNA作为在步骤4.3存储在-20或-80℃的样品，如果需要长期存储。 </li></ol></li></ol> 6。 DNA的表征焦磷酸测序<ol><li>确保未标记的本机（12℃）的DNA占据密度范围从1.705克/ ml至1.720克/毫升，而13 C-标记的DNA具有约1.720-1.735克/毫升的密度。请注意，无论是原生和13 C标记的DNA从环境样品中提取出可以跨越几个梯度组分。预计光的DNA能与组分9-11和沉重的DNA能与分数相关的4-6关联。 </li><li>结合的关键部分来创建一个单一的"编译"部分代表交流盘带的密度分辨DNA的特定峰值分配。可选地，其它方法使用同位素比率质谱仪（IRMS）22或一个指纹识别方法，如变性梯度凝胶电泳（DGGE）23，检查分离的馏分可以帮助测序之前选择合适的馏分。 </li><li>进行的PCR扩增的细菌的16S rRNA从各个梯度的编译重，中，轻馏分基因焦磷酸测序。在SIP中使用这种方法，鉴定血统和代谢活性的细菌物种相对多度孵育样品是可能的。如下进行焦磷酸测序: <ol><li>使用罗氏454 FLX仪器与试剂钛进行扩增焦磷酸测序。 </li><li> （ - GAGTTTGATCNTGGCTCAG -3 5）和Gray519r（5 - GTNTTACNGCGGCKGCTG -3）24,25扩展与日通过融合引物Gray28F准备条形码的扩增子的复E中各个底漆适配器A / B和样品特异性多重标识符（MID）。 </li><li>应用1步PCR用30个循环，使用热启动Taq DNA聚合酶，以产生测序文库。 </li><li>序列基于该供应商协议的扩增子，并延长读取从前进方向（Gray28F）上述<a href="http://www.researchandtesting.com/">http://www.researchandtesting.com/</a> 。 </li></ol></li></ol> 7。焦磷酸测序数据分析<ol><li>分析原始序列读取由454焦磷酸测序与"定量见解微生物生态学"，QIIME软件管道26产生的。按照如下步骤进行处理: <ol><li>最初，转换454-机器产生的SFF文件，FASTA，QUAL和Flowgram文件与process_sff.py脚本。 </li><li>验证映射文件（基于check_id_map.py）和多路分配读生物SAM普莱斯的split_libraries.py脚本。修剪为50的滑动窗口的大小和25的平均质量的截止参数的低质量的结束，也消除了数据集中暧昧的短序列（长度&lt;200个基点）。 </li><li>使用denoise_wrapper.py去噪的454数据。 </li><li>接下来，使用多个OTU采摘方法（pick_otus.py与97％的序列相似性边界品位）群集高品质读入操作分类单元（OTU单板）。 </li><li>使用pick_rep_set.py从每个OTU挑选有代表性的序列。 </li><li>指定分类的基础上assign_taxonomy.py代表序列集，使用核糖体数据库计划（RDP）的分类与最小置信度来记录任务设置为0.80。 </li><li>对齐代表序列对预校准Greengenes核心16S序列，使用该算法PyNast（align_seqs.py用最小的序列同一性百分比设定为75）。 </li><li>此外，通过运行identify_chimeric_seqs.py消耗嵌合体从进一步的分析。 </li><li>删除所有空白位置之前建立的系统发育树（make_phylogeny.py）（用于系统发育推论没有用处）与lanemask模板（filter_alignment.py）。 </li><li>最后，make_otu_table.py生成OTU表，描述每个样本内的细菌种系型的发生。使用OTU表构造热图用于比较的细菌数量和活性。 </li><li>如果需要，计算α和β多样性和内部之间的样本，分别。以同样的方式，稀疏曲线（多样性与测序深度的曲线图），并代表微生物群落之间的关系主要坐标分析（后交通动脉）图也可以制备。 </li></ol></li></ol>

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H., Conrad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+stable+isotope+probing+of+methanogenic+archaea+in+the+rice+rhizosphere.">In situ stable isotope probing of methanogenic archaea in the rice rhizosphere.</a> Science. 309, 1088-1090 (2005).</li><li>Radajewski, S., Ineson, P., Parekh, N. R., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+probing+as+a+tool+in+microbial+ecology.">Stable-isotope probing as a tool in microbial ecology.</a> Nature. 403, 646-649 (2000).</li><li>Lueders, T., Pommerenke, B., Friedrich, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+of+DNA-+and+rRNA-based+stable+isotope+probing+by+fractionation+and+quantitative+analysis+of+isopycnic+centrifugation+gradients.">Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.</a> Environ. Microbiol. 6, 73-78 (2004).</li><li>Ishak, H. D., 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=Bacterial+diversity+in+Solenopsis+invicta+and+Solenopsis+geminata+ant+colonies+characterized+by+16S+amplicon+454+pyrosequencing.">Bacterial diversity in Solenopsis invicta and Solenopsis geminata ant colonies characterized by 16S amplicon 454 pyrosequencing.</a> Microb. Ecol. 61, 821-831 (2011).</li><li>Sun, Y., Wolcott, R. D., Dowd, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tag-encoded+FLX+amplicon+pyrosequencing+for+the+elucidation+of+microbial+and+functional+gene+diversity+in+any+environment.">Tag-encoded FLX amplicon pyrosequencing for the elucidation of microbial and functional gene diversity in any environment.</a> Methods Mol. Biol. 733, 129-141 (2011).</li><li>Kuczynski, 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=Using+QIIME+to+Analyze+16S+rRNA+Gene+sequences+from+microbial+communities.">Using QIIME to Analyze 16S rRNA Gene sequences from microbial communities.</a> Curr. Protoc. Microbiol. Chapter 1, Unit1E 5 (2012).</li><li>Dillon, R. J., Dillon, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+bacteria+of+insects:+nonpathogenic+interactions.">The gut bacteria of insects: nonpathogenic interactions.</a> Annu. Rev. Entomol. 49, 71-92 (2004).</li><li>Neufeld, J. D., 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=DNA+stable-isotope+probing.">DNA stable-isotope probing.</a> Nat. Protoc. 2, 860-866 (2007).</li><li>Dumont, M. G., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+probing+-+linking+microbial+identity+to+function.">Stable isotope probing - linking microbial identity to function.</a> Nat. Rev. Microbiol. 3, 499-504 (2005).</li></ol>

我们什么都没有透露。

大多数昆虫的肠道中藏着丰富而复杂的微生物群落，通常为10 7 -10 9原核细胞有提出，在大多数情况下，数量上超过了宿主自身的细胞。因此，昆虫肠道是一个"热点"不同微生物的活动，即微生物关系的多个方面，从发病到预留共生27。虽然许多研究都说明一个惊人的各种昆虫肠道微生物群落，肠道菌群的代谢活动的特征是罕见的。稳定同位素探测方法现在提供了一个可能性，区分复杂的菌群背景的独立种植，甚至在体内的活跃成员。我们应用这个宝贵的工具，研究肠道菌群在昆虫模型系统，棉叶虫（ 海灰翅夜蛾 ）。由于葡萄糖是在斜纹夜蛾肠的主要糖分，在这个演示中，我们喂拉尔VAE用人工饲料掺入的13 C-葡萄糖额外的，但生理剂量的跟踪新陈代谢活性细菌在肠道菌群。与天然葡萄糖建立一个相同的培养提供了一个临界控制用于随后的比较，以确保核酸的任何明显的标记并不是方法本身的工件，例如在DNA有助于分离高G+ C含量。 在葡萄糖浓度原位不久还减少了实验偏差。相关的严格的实验设置的DNA-SIP研究的其他方面的考虑已在别处28,29讨论。肠道细菌通常是代谢非常活跃，有短代时间相比，大多数陆地和水生环境中的微生物。因此，一个24小时连续进料已经造成了显著的标签。注意，大量的宿主的基因组DNA也从肠道组织中，这应该被考虑到共萃取讨论结果时考虑。 典型的梯度级分的表征依赖于低分辨率指纹的方法，如末端限制性片段长度多态性（T-RFLP），且耗时克隆文库构建链接的数据。它只是在最近，高通量的第二代测序技术的出现，可以快速，具成本效益，并详细复杂的微生物群落的分析。在这里，我们应用这种新技术来直接测量密度梯度组分的遗传组成，并确定代谢活性的细菌，这给了增强的灵敏度与凝胶电泳为基础的方法相比，促进了随后的系统发育分类。由两个13 C标记和12架 C-对照样本比较相当于重馏分中，我们发现， 成团泛菌和肠球菌成为标记后同位素探测。一旦代谢olically活性细菌已经确定，其他的分析，例如使用特异性探针与标记的DNA的宏基因组分析，从活跃的社区成员恢复原位杂交荧光（FISH）技术，可进行提供一个全面的洞察与主机相关联的真实共生体。基于这里所建立的系统上，其它的13 C-标记的碳源也可以送入主机解剖涉及靶向的肠道代谢途径中的细菌，例如，标记的纤维素鉴定参与宿主消化过程中活性的细菌。用这种新方法的发展，昆虫肠道微生物的作用将变得比现在更为明显。 总的来说，这里描述的方案表示用于确定肠道菌群，其中还可以应用于评估瑾珑的具体作用的代谢活动以简单，快速和有效的方法现实共生体在寄主营养，排毒和防御。

昆虫细菌共生是已知的昆虫物种7的大量出现。在这些共生微生物中发挥重要作用的昆虫的生长和发育。微生物已被证明有助于昆虫繁殖1，信息素生物合成3，营养，包括必需氨基酸4的合成中，并且不可访问的食物消化到主机。尽管庞大的各种肠道细菌性协会，更不用说有人知道他们赞成昆虫发挥职能作用。仅在发生了白蚁，木素纤维素的共生消化进行的原核生物，原生动物和真菌，已被广泛研究8,9。与此相反，鲜为人知的是，目前在通才昆虫的肠道即斜纹夜蛾的共生体， 海灰翅夜蛾，而且由于其频繁的植物宿主的转变，一般北京时间昆虫及其相关的肠道细菌群落被永久暴露在与他们的饮食习惯食用的植物有大量的植物化学物质的新的挑战。除了 ​​这一点，在肠道环境中鳞翅目，指本身为细菌的生长，因为高肠道pH值10的恶劣环境。特别是在S的情况下北沙参 ，它的范围从10.5前肠，中肠约 9至近7在后肠11 pH值。另一方面，细菌群落与S的肠道相关北沙参很简单。唐，Freitak， 等 12报道最多36种系型属于共有7种不同的细菌种类与这种昆虫相关的细菌群落的唯一成员。除此之外，需要的昆虫生长在实验室中没有复杂的饲养过程。此外，这和昆虫的生命周期短便于多克enerational研究，把这个物种转化为研究肠道微生物相互作用的理想模型系统。 用基于PCR的测序技术的出现，研究处理的一些生物体（ 即人，昆虫或海洋生物）肠道生物群的数目已经增加。此外，结果是独立的分离培养肠道窝藏细菌像过去那样。几乎99％的细菌是不耕地和当时在肠道中的环境条件的模拟是困难12。通过使用PCR，16S rRNA基因片段（细菌中广泛使用的系统发育基因标记物）可以被选择性地从肠道细菌群落，测序的混合DNA模板扩增，并克隆。有了这些信息，用户可以从公共数据库13,14中检索的序列信息后，以确定细菌种类。然而，测序方法来描述细菌社区仍然不足，由于缺乏对社区内的个别品种的内在代谢贡献的信息。 稳定同位素探测（SIP）是一种很有前途的文化自由技术。它通常用于在环境微生物学分析链接到特定代谢活性的微生物的系统发育。这是通过跟踪稳定同位素标记的底物从原子到微生物的生物标志物，如磷脂衍生的脂肪酸，DNA和RNA的5实现的。当考虑核酸，该方法是基于对来自13采用密度梯度离心6的未标记的DNA C标记的DNA或RNA的分离。由于该DNA标签和代谢活性之间的这种直接连接，所述核酸的下游分子分析确定的物种，并提供有关的代谢活动的信息。此外，DNA-SIP和焦磷酸测序技术相结合而适用的Pilloni，冯内策等人 ，15，允许一个特定的简单和灵敏的鉴定细菌种类的存在于重的13 C-标记的DNA级分。到现在为止，这种技术已被应用到描述有氧16,17和厌氧条件下18,19涉及在土壤中生物地球化学过程中的细菌群落。除了 ​​在环境科学的使用，该技术已在医学应用所报告的雷查德等人的 5，谁描述响应于不易消化的碳水化合物人类肠道菌群的不同进化群体的代谢活动。 这里，我们使用13 C-葡萄糖到"标签"的代谢活性细菌种类在肠道中的DNA。葡萄糖是由大多数的细菌种类以及广泛恩特纳-Doudoroff（ED）途径利用，虽然异常被称为20糖。这证明利用13 C-葡萄糖作为一种可靠的代谢探针，提供感兴趣的代谢物，并沿既定途径碳源之间的链接。取决于科学问题，其它底物， 即 13 C-甲烷，13 CO 2，或13 CO 2的气氛下提出的植物，可以用来解决代谢活动。 在这一点上，我们提出了一个多面手的昆虫，即S的肠道细菌群落的代谢特性应用协议北沙参 （ 鳞翅目，夜蛾科 ）。此外，该技术被耦合到的焦磷酸测序，从而允许昆虫肠细菌群落的鉴定具有高的分辨率和精度。作为主基板，13 C-标记的葡萄糖在实验期间使用。

<table border="0" cellpadding="0" cellspacing="0" width="872"> 
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dumont #5 Mirror Finish Forceps</td>
			<td style="width:203px;">Fine Science Tools</td>
			<td style="width:135px;">11251-23</td>
			<td style="width:151px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Speed Vacuum Concentrator 5301</td>
			<td>Eppendorf Germany</td>
			<td>5305 000.304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic pestle</td>
			<td>Carl Roth GmbH Co. Germany</td>
			<td>P986.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoVue spectrophotometer</td>
			<td>GE HealthCare, UK</td>
			<td>28-9569-58</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mastercycler pro/thermocycler</td>
			<td>Eppendorf Germany</td>
			<td>6321 000.515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agagel Standard Horizontal Gel Electrophoresis chamber</td>
			<td>Biometra</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge (Optima L-90K)</td>
			<td>Beckman</td>
			<td>A20684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge rotor (NVT 90)</td>
			<td>Beckman</td>
			<td>362752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC pump</td>
			<td>Agilent</td>
			<td>1100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quick-Seal, Polyallomer tube</td>
			<td>Beckman</td>
			<td>342412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transilluminator UVstar 15 </td>
			<td>Biometra</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of metabolically active bacteria in the gut of the generalist spodoptera littoralis via dna stable isotope probing using 13c-glucose

大多数昆虫的内脏是由致病性共生细菌的复杂社区居住。在这样的微生物群落能够识别共生或互利共生菌物种。后者的，已经观察到具有多种功能的昆虫， 即有助于在昆虫繁殖1，增强免疫反应2中 ​​，信息素产生3，以及营养，包括必需氨基酸4的合成，等等。 由于这些协会的重要性，已经做了很多努力，社区表征下至各个成员。然而，大多数这些努力的依据是栽培方式或倚赖其测序的最终鉴定16S rRNA基因片段的产生。不幸的是，这些方法只能识别存在于肠道细菌种类和没有提供了信息离子对微生物的代谢活性。 在昆虫的内脏表征代谢活性的细菌种类中，我们使用稳定同位素体内采用13 C-葡萄糖作为底物的通用探针（SIP）。这是一种很有前途的文化自由的技术，使微生物系统发育的联动，其特定的代谢活动。这是可能的，通过跟踪稳定同位素标记的底物从原子到微生物的生物标记物，如DNA和RNA 5。 13 C中的同位素掺入到DNA中增加了标记的DNA的密度相比于未标记的（12℃）1。在结束时，13 C-标记的DNA或RNA是通过密度梯度超速离心法从12 C-未标记的类似1 6分离。分离的核酸同位素的后续分子分析提供了种代谢活性和身份之间的连接。 在这里，我们提出用在一个多面手昆虫（我们的模型系统）， 斜纹夜蛾 （ 鳞翅目，夜蛾科 ）的肠道表征代谢活性细菌的协议。做了DNA的系统发育分析采用焦磷酸测序技术，这使得高分辨率和精确度在昆虫肠道细菌群落的识别。作为主基板，13 C-标记的葡萄糖被用在实验中。将基板输送到使用人工饲料的昆虫。

实现存在于昆虫肠道的代谢活性细菌的足够标识，昆虫必须暴露在为先前的优化期间足以使标记的较重的馏分的分离容易地从非标记打火机1的13 C-富含衬底。在我们的案例中，13 C-葡萄糖在10毫米1天（ 图1A）的终浓度在补充人工饲料。在人工饲料控制昆虫提供了正常葡萄糖（ 图1B）的相同。如前面提到的，该DNA组分分离（从打火机较重）是整个过程的基础。因此，要执行的第一个步骤是从所关注的组织DNA的提取，在这种情况下，该细菌群落与昆虫肠相关联。以确认其质量来运行凝胶电泳，以确定是否从ANALY得到在所有的任何DNA是非常重要捷思组织。在图1C中，它是可观察的图像，确认阳性DNA提取的顶部基因组DNA的强条带。此外，有必要进行诊断性PCR，用细菌的通用引物，以确定细菌的DNA（ 图1D）的存在。该肯定结果是重要的，以决定是否还继续与所提取的宏基因组DNA的分离。 超速离心后，一旦在每个分离的馏分中的DNA的量被确认，和密度的测量完成时，绘制的平均分数密度以g / ml的多级分编号，以确认正确的梯度形成的管，它通常包括一从1.690（分数为12或11）1.760克/毫升克/毫升的密度范围 （分数为1）。定量焦磷酸测序直接进行对代表的SIP梯度揭示物种谱系和相对丰度（ 图2 </strong&gt;的）。在这里，我们测序同时从13 C-葡萄糖修正样本和未标记的控制，这有助于在社会剖析活跃的人口中重质馏分。测序后，共120045高质量读取与生成的404个核苷酸的平均长度。在焦磷酸测序更新显示某些物种的一种增加的丰度，包括肠球菌属和泛菌属的标记的样品在（13 C-标记的DNA， 图3）与对照组（12 C控制的DNA， 图3）相比较。因此，这些细菌被认为是代谢活跃，并值得进一步研究。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50734/50734fig1.jpg" width="500px" /> 图1 13 C-葡萄糖喂养实验，DNA提取及细菌16S rRNA基因根的PCR扩增<html> Ë。 （A）13 C-葡萄糖修订人工饲料用甜菜消耗北沙参幼虫（B）原生血糖（12 C-葡萄糖）修订人工饲料从同一批次中（A）中使用的昆虫，它作为控制消耗的幼虫从幼虫在13 C-葡萄糖治疗组和12架 C-葡萄糖的对照组肠道组织。（C）典型的宏基因组DNA的提取物。三个生物学重复（泳道1,2和3）被包含在每个组中。 M代表1kb的DNA标记物（D）的PCR扩增用万能细菌的引物组通过使用所提取的宏基因组DNA作为模板生成正确的1.5 kb的16S rRNA基因产物。泳道1为PCR阳性对照。泳道2和3表示的13 C-葡萄糖处理的样品和12 C-葡萄糖对照。<html> JPG"目标="_blank"&gt;点击这里查看大图。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50734/50734fig2.jpg" width="600px" /> 图2。火焰兵-SIP实验涉及氯化铯密度梯度离心和组分特性与分离的DNA的焦磷酸测序的轮廓H =较高的丰度，L =低丰度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50734/50734fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50734/50734fig3.jpg" width="500px" /> 图3。细菌的多样性和相对丰度在天然葡萄糖饲喂幼虫（12 C-对照DNA）和13 C-葡萄糖饲喂幼虫（13 C-标记的DNA）的重馏分。/ www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50734/50734fig3highres.jpg"目标="_blank"&gt;点击这里查看大图。

Watch this Scientific Journal Video about Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose at JoVE.co

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

与海灰翅夜蛾的肠道相关活动的细菌群落，由稳定同位素探测（SIP）耦合到焦磷酸测序确定。使用这种方法，识别社区内的代谢活性细菌种类与高的分辨率和精度完成。

的代谢活性细菌在通才的肠道鉴定<em&gt;海灰翅夜蛾</em&gt;通过DNA的稳定同位素探测使用<sup&gt; 13</sup&gt; C-葡萄糖

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

identification-metabolically-active-bacteria-gut-generalist

Nanyang Technological University

Research

JoVE Journal

Biology

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

15.3K 次观看.  Max Planck Institute for Chemical Ecology. 与海灰翅夜蛾的肠道相关活动的细菌群落，由稳定同位素探测（SIP）耦合到焦磷酸测序确定。使用这种方法，识别社区内的代谢活性细菌种类与高的分辨率和精度完成。 

视频: Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

从白蚁肠道微生物（Zootermopsis Angusticollis）中提取的DNA和可视化肠道微生物

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

科研

生物学

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA的稳定同位素探测（DNA的SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

使用可视化的DNA复制DNA纤维的技术，在脊椎动物的模型系统DT40

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

的代谢活性细菌在通才的肠道鉴定

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