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

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...

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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?...

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。

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	<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...

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 ...

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

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

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

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

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

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

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

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

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

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

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

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

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

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

建立稳定的双生糖核糖核关系从口腔

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...