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行测定。 </li><li>找出一个替代剪接制度，应该感兴趣的蛋白定位到正确的细胞器。对于这项工作，剪接位点作为一个自然拟南芥系统6观察。 mRNA序列进行了重新设计留下天然剪接位点，但序列置于编码叶绿体或peroxis青梅靶向的交替剪接内含子内标签（ 图1）。 </li></ol> 2，吉布森DNA的组装部件参见图2。 吉布森组装方法在预制的组合，以1取决于几种酶的作用）部分地消化双链DNA的末端，使单链和2）退火免费碱基对的DNA邻近的部位。这允许DNA片段的克隆没有限制酶切位点的限制。 <ol><li>选择用于在该DNA的质粒构建体中的元素的顺序。作为一个例子TriTag-1具有的要素：a）一种质粒载体（孔，含有已知在植物中卡那霉素抗性标记的工作GFP的构建体，和复制的大肠杆菌和根癌土壤杆菌宿主），b）一种起源启动子序列，（P ENTCUP2，证明是组成型植物），三）靶向序列（CTS）的叶绿体，c）一种过氧化物酶体靶向序列（PTS），以及d）如前面6描述了一系列的剪接位点。 </li><li>设计构建基为基带，在硅片设计软件。 APE是一个开放源码的免费软件包对这项工作非常有用。 注：TriTag-1，具有顺序为：质粒载体，启动子，ATG，剪接供体位点，叶绿体靶序列，剪接供体位点，中性的网站，剪接受体位点，过氧化物酶体靶序列，剪接受体位点，绿色荧光蛋白为包含在质粒载体。 <ol><li>设计在硅片 DNA结构，使得订货引物进行PCR时，有每片之间有50 bp的重叠。它可以使用50个碱基的引物：25 bp的每个方向。 <ol><li>一定要保持每个序列块的起源轨道。它是为：质粒进行种植和微量制备;一个线性序列，可被用作PCR的模板;或一个序列，将需要合成的商业？有几家公司为片段&lt;500 bp的提供低成本DNA合成服务。在这种情况下，TriTag本身是437碱基所以这是有利的，商业上订。 </li><li>最好的结果会来，如果所有的片段的PCR扩增，并从它们的模板DNA进行纯化。抗性标记是一个好地方来划分这个大的DNA成两个较小的模板更容易PCR扩增的。 </li><li>限制限制性内切酶切割DPNI甲基化DNA。如果PCR的模板是从E.一个小量制备大肠杆菌，DPNI处理将去除污染的DNA模板。 </li></ol></li></ol></li><li>单独净化所有的DNA序列和洗脱水，TE或缓冲的EB。 <ol><li>孔载体部分1，〜3,000 bp的（绿色箭头）。 PCR扩增并进行DPNI处理。 </li><li>孔载体部分2，〜3,000 bp的（黑色箭头）。 PCR扩增并进行DPNI处理。 </li><li> TriTag插入，437个基点（蓝色箭头）。商业订购。 Řesuspend在DDH 2 O。 </li><li> P ENTCUP子，〜750个基点（橙色箭头）。 PCR扩增，并进行DPNI处理。 </li></ol></li><li>测量各DNA片段的浓度。瞄准150纳克/微升向量件。 </li><li>取吉布森组件组合的等分试样出冰箱和解冻冰上。这是市售的，而且简单，使在实验室12-14。 <ol><li>有两个步骤来此配方：一种粘稠的5倍预混液和15μl的反应大小的1.33倍等分。的5倍主混合物含有：3毫升的1M的Tris-HCl pH为7.5，加入300μl的1M MgCl 2的，每种dNTP各600微升的10mM，300微升的1M DTT，加入1.5g PEG-8000，20毫克NAD和双蒸0到6毫升准备320微升等分（18），并冻结所有，但一个在-20°C。该解决方案将非常粘稠。标注这些“5X等温线的缓冲区” </li><li>要剩下的一个（320微升），加1.2μLT5核酸外切酶，20微升的Phusion高-FIDelity DNA聚合酶，160微升的Taq DNA连接酶和700微升双蒸2 O。制备15微升等分试样（〜80），在冰上在PCR管中并储存于-20℃。 </li></ol></li><li>确定卷的每个片段的等摩尔量。有几个在线计算器，包括Gibthon.org。应该有一个总的5微升所有DNA碎片。将它们添加到15μL吉布森混合等分。如果这需要体积太小，移液管，稀释的DNA片段和/或使用一个以上的等分试样。 <ol><li>不要忘了准备单独的载体DNA的控制（ 如含有抗生素抗性标记的DNA的一部分），并与水的体积。 </li></ol></li><li>孵育在热循环仪的管在50-55℃30-60分钟。装上冰和转化为E。通过热休克化学感受态细胞大肠杆菌 。不要使用电感受态细胞。在高盐浓度和相对低的DNA浓度可能导致细胞电弧。 <ol><li>适用于所有20μl到一个商业的200微升氯化钙制备感受态大肠杆菌 。 在冰上孵育30分钟，热休克60秒，在42℃下</li><li>返回到冰2分钟，应用750微升SOC或LB培养基中，并回收摇动在微量离心管30分钟，在37℃下板放在LB +阚（或其它适当的抗生素）的混合物中，并适当稀释。这可能导致较高的背景（和一些非靶重组事件更高）比传统的结扎的克隆，但它是值得的，筛选大约10个菌落。 </li></ol></li><li>通过序列确认克隆和存储的等分试样在-80℃下</li></ol> 3，基因枪法烟草的转染与基因枪这是一种建立在朱庇特的18,19技术。关键步骤和差异如下所述。 <ol><li>增长50-100毫升大肠杆菌合作Li或200毫升农杆菌 。马克西 - 预习文化。约1500纳克/ A浓度微升才能继续。 </li><li>制备基因枪子弹如前所述。它们加载到基因枪。 </li><li>对于本氏烟烟叶表皮组织的转染，选择大叶近3个月的旧厂房的基础。小心切开它，并把它底部朝上湿纸巾15厘米的培养皿中。 </li><li>的压力，他对基因枪设置为200-250磅。 </li><li>仔细瞄准基因枪肋骨之间在叶的底部，约3-5厘米以上吧。释放安全和递送颗粒的叶。每个子弹在叶上的同一位置，可以使用两次。如果叶爆炸，丢弃，并开始新的一页，有叶韧性一些差异，由于生长条件如年龄，水分等。对于一个12厘米的叶片，期望以适合约6张。 </li><li>存储叶子，覆盖着一顶培养皿在板凳上弱光2-3天。 </li><li>检查配备了紫外荧光解剖显微镜的叶子，然后搜索表达GFP单个细胞。剖析叶5-10毫米的部分。 </li><li>淹没在叶下〜200微升0.1％的Triton X-100深井显微镜幻灯片。洗涤剂有助于防止气泡的表面上形成，且深井泵显微镜载玻片允许更大的组织成像区域。 </li></ol> 4，激光共聚焦转染组织的显微镜这些指令各有不同，每一个仪器，所以它必须得到适当的培训。 <ol><li>用于荧光检测的实验中，激发激光器设置为489 nm，而设置在光电倍增管探测器500-569纳米的GFP荧光和630-700 nm的叶绿素自发荧光。用40倍水浸泡的目的细胞图像。 </li></ol>

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Vis. Exp. (12), (2008).</li><li>Hollender, C. A., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+Fluorescence+Complementation+(BiFC)+Assay+for+Protein-Protein+Interaction+in+Onion+Cells+Using+the+Helios+Gene.">Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene.</a> (40), (2010).</li><li>Li, M. Z., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+homologous+recombination+in+vitro+to+generate+recombinant+DNA+via+SLIC.">Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.</a> Nature Methods. 4 (3), 251-256 (2007).</li></ol>

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 ...

transient-gene-expression-tobacco-using-gibson-assembly-gene

Research

JoVE Journal

Environment

20.4K 次观看.  Harvard University. 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。 

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

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

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

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 exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

成绩单及代谢物谱烟草树和杨树的评价作为原料的生物基工业

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

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

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

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

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

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 factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

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

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

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

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

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

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

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

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

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 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 the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

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

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

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

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

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

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 describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

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 ...