Name: transient gene expression in tobacco using gibson assembly and the gene gun
Uploaded: 2014-04-18T18:30:00
Description: Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

作者要感谢马萨诸塞州总医院的仁为辛本氏烟苗的慷慨捐赠。仁布什帮助我们大大的意见，在植物生长，并设立一个生长室面积。韦斯研究所的汤姆·费兰特共聚焦显微镜提供了重要的帮助。作者要特别感谢波士顿儿童医院唐Ingber和威斯研究所基因枪和相关耗材的慷慨捐赠。资助这项计划是通过与能源高级研究计划署的署（ARPA-E奖＃DE-000079）为PAS，JCW，和MDM的合作协议规定，并通过Chimerion生物技术公司的MJV。

的或者拼接序列的多细胞器靶向1。设计<ol><li>确定网站最终蛋白的表达。对于这项工作，兴趣是在瞄准的叶绿体，过氧化物酶体和细胞质。 </li><li>使用该文献以确定已知的靶蛋白与感兴趣的细胞器的蛋白质和DNA序列。在这种情况下，叶绿体靶向序列来自拟南芥的L-isoaspartate甲基6,15，和来自拟南芥转甲状腺素状的S-尿囊素合酶16,17靶向序列的过氧化物酶进...

<ol>
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在这项研究中，简单的策略描述了用于定位的单个转基因蛋白在植物中多种细胞区室。我们的目标是设计构造，它会表达一个单一的基因在烟草本塞姆氏多个细胞器。策略包括合理的设计基于GFP的DNA构建体，吉布森组装，交付质粒叶细胞与基因枪，并观察其结果与激光共聚焦显微镜。 三种不同的短，N-末端标签被设计为同时叶绿体，过氧化物酶体和胞质溶胶定位11?...

这项工作是代谢工程/合成生物学项目，其中植物细胞工程表达报告蛋白在多种细胞器，但只有一个单一的DNA结构。 一种方法对目标蛋白以多个位置包括克隆多个基因拷贝，每个包含一个不同的本地化的肽。每个副本都必须通过连续的重转换被引入，或者，通过回交单转换1。这涉及到额外的克隆，并通过每总站1定位标记的限制。 另一种方式来定位一个蛋白到多个位置是通过选择性剪接2-5。 RNA是从一个单一的基因转录，但转录物的不同副本中每细胞多于一个的方式处理不同，往往。这可能会导致在从一个单一的基因转录的细胞多于一个信使RNA。这些不同的信使Ř的RNA可编码为同一蛋白质的不同的同种型，或在一个移码，不同的蛋白质完全的情况。虽然选择性剪接在文献中已经描述了许多年，操作和保守的供体和受体剪接位点的机制仅被阐明，最近6。由于这些网站都被更好地描述，他们打开了工程的机会。 在多个细胞器表达一种蛋白质，当植物代谢的工程师都面临着一个挑战。为一种蛋白质，是将其定位于一个以上的细胞器中，工程师必须克隆同一基因多次，每一个独立的信号序列，它引导到感兴趣的细胞器。在三个细胞器的单个基因，这简直就是三个基因。但是，对于一个六基因代谢途径，这种扩展到18个基因，一个显著克隆的努力。结合多种定位序列成一个单一的，或者拼接基因标志ificantly减少了这方面的努力。例如，重新设计光呼吸7,8和类异戊二烯合成9,10同时涉及叶绿体和过氧化物酶体。在我们的例子中，我们利用了剪接位点作为一个自然拟南芥系统观察前面所述6。我们合理地重新设计的mRNA序列而使天然剪接位点单，但放置顺序，将在交替剪接的内含子（ 图1）内的编码叶绿体或过氧化物酶体靶向标记。所表达的蛋白质可能有或可能没有一个标签，这取决于前体mRNA编码它是否被切除的内含子（ 图1g和1小时 ）。就在这项工作中提出的结构的设计的更多信息，请参阅本文的姊妹篇11。 因为这仍是一个显著克隆的努力，吉布森组装，克隆DNA结构的新方法，为used的建设。可以使用吉布森方法对于任何序列，无论限制性位点（ 图2）12-14。酶的特定组合允许单步​​，等温组件。在该方法中，一些双链线性DNA部分被设计为使得它们具有的〜50bp的重叠序列。吉布森组装酶混合物部分消化线性DNA部分，露出的单链同源序列。这些部分单链的序列被重新​​退火的反应混合物中，导致快速的一步，顺序无关的，结扎无亚克隆反应。 这项工作说明1）合理设计选择性剪接表达在植物叶绿体，过氧化物酶和细胞液的结构，2）他们的克隆使用吉布森组件的新结扎-free方法，3）其输送到烟叶细胞与基因枪，和4）结果显示细胞器的定位，与绿色荧光蛋白观察和共聚焦显微镜。

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligonucleotide primers</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td style="width:167px;">Design specifically for construct</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneBlocks</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td>500 bp oligonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ApE software</td>
			<td style="width:191px;">U Utah</td>
			<td style="width:119px;">(download)</td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MinElute kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">28004</td>
			<td>Used to purify PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep spin miniprep kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Used to prepare cloning-appropriate amounts of plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Master Mix with GC Buffer</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0532S</td>
			<td>Used to PCR-amplify gene of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibson assembly reaction mix</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">E2611L</td>
			<td>Master mix of the following 9 ingredients</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M Tris-HCl pH7.5</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:119px;">T1075</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M MgCl2</td>
			<td style="width:191px;">G Biosiences</td>
			<td style="width:119px;">82023-086</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP mix</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0192</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M DTT</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0861</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG-8000</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">19966</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:119px;">A1124,0005</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T5 exonuclease</td>
			<td style="width:191px;">Epicentre</td>
			<td style="width:119px;">T5E4111K</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">F530S</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Taq DNA ligase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0208L</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibthon.org</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>Website to simplify calculations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasmid PLUS Maxi kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">12963</td>
			<td>Used to prepare DNA for gene gun bullets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Gun system</td>
			<td style="width:191px;">BioRad</td>
			<td style="width:119px;">165-2451</td>
			<td>Includes all parts necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nicotania benthamiana</td>
			<td style="width:191px;">(n/a)</td>
			<td style="width:119px;">(n/a)</td>
			<td>Gift of Jen Sheen, MGH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:191px;">Leica</td>
			<td style="width:119px;">SP5 X MP</td>
			<td>Imaging of resultant cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deep well slides</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71561-01</td>
			<td>Used for confocal imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">A Plasmid Editor (ApE) </td>
			<td style="width:191px;">University of Utah </td>
			<td style="width:119px;"></td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gibthon:Ligation calculator</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>http://django.gibthon.org/tools/ligcalc/</td>
		</tr>
	</tbody>
</table>

transient gene expression in tobacco using gibson assembly and the gene gun

为了针对单一蛋白质多亚细胞器，植物通常复制有关的基因，并使用复杂的监管策略，包括不同的启动子和/或信号序列分别表达各自的基因。代谢工程和感兴趣的目标酶到特定的细胞器合成生物学家都面临着一个挑战：对于一种蛋白质，是被定位于一个以上的细胞器，工程师必须复制相同的基因多次。这项工作提出了解决这一策略：利用mRNA的选择性剪接。这项技术需要建立叶绿体和过氧化物酶体靶向序列的优势，将它们组合成一个单一的mRNA被选择性剪接。一些剪接变体将被发送到叶绿体，一些对过氧化物酶，以及一些到细胞质。这里该系统被设计用于多细胞器靶向与可变剪接。在这项工作中，GFP预计将EXPRessed在叶绿体，细胞质和过氧化物酶体通过一系列合理设计5&#39;基因标签。这些标记必须减少当异源基因需要被表达于多种细胞内的细胞器需要克隆的量的电位。该构建体被设计成在先前的工作11，以及使用吉布森装配，不需要限制酶结扎独立克隆方法进行克隆。将得到的质粒导入本氏烟草叶表皮细胞与改性基因枪协议。最后，叶变换观察与激光共聚焦显微镜。

设计工作是显著规划的结果。小说这个项目是利用选择性剪接来创建一个前体mRNA被翻译成差异表达蛋白。这些蛋白被表达在不同的细胞器，在这种情况下，叶绿体，过氧化物酶体和/或细胞质中。我们适于天然拟南芥基因被选择性剪接6，并置于已知叶绿体6和过氧化物酶体17定位在备用的外显子序列（TriTag-1和TriTag-2）。 TriTag-3由嵌入在叶绿体序列的低复杂性区域中的...

Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

这个工作描述了一种新颖的方法，用于选择性地靶向亚细胞器在植物中，使用BioRad公司基因枪进行测定。

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

科研

环境科学

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

跟踪微生物污染在零售环境中使用荧光粉 - 零售熟食店环境范例

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

重组纤维素酶Cel5A从表达<em&gt;里氏木霉</em&gt;在烟草

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

食品筛查为1类整合和基因盒

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

黄曲霉毒素在花生RNAi介导的控制：方法分析霉菌毒素的产生和转基因表达的花生/<em&gt;黑</em&gt;病害系统

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...