感谢麦克拉厅，拉里萨Bispo的古斯塔沃和塔瓦雷斯的技术援助。

那些生吃或熟轻食物是最关注的问题对人类的健康。例子包括蔬菜沙拉，水果，贝壳类和甲壳类动物。 1.样品采集<ol><li>采集样品，最大限度地减少污染条件下，在运输过程中保存在单独的，干净的袋子。一旦收集，样品应当存放在4℃和24小时内处理。 </li></ol> 2.富集培养制备<ol><li>水果和蔬菜： <ol><li>放置约10g材料在耐用的塑料袋中。如果在处理较大的材料，集中在水果或蔬菜的皮肤上。添加90毫升0.1M磷酸钠缓冲液，pH 7.0和过程中桨式搅拌器30秒。可替代地，该材料可手动搅动如果桨式搅拌器不可用。 </li><li>收集液体倒入两个50毫升猎鹰管。离心管在4000 XG 7分钟沉淀细菌。 </l我&gt; <li>小心取出上清液，在30ml无菌卢里亚BERTANI（LB）肉汤暂停沉淀细菌。 </li><li>孵育在振荡器上O / N，保持一个管在25℃，另一个在37℃。文化应在每分钟200振荡（OPM）被动摇。商店培养在4℃下直到需要。 </li></ol></li><li>海鲜： <ol><li>解剖出胃（牡蛎）或消化道（虾），使用无菌镊子和镊子，并放入无菌的1.5mL管中含有200微升的0.1M磷酸钠缓冲液，pH 7.0。浸渍样品以创建匀浆。 </li><li>分配5毫升LB肉汤分成两无菌5毫升管，并与海鲜匀浆接种每个。 </li><li>孵育在振荡器上O / N，保持一个管在25℃，而另一个37℃。文化应该在200 OPM动摇。商店培养在4℃下直到需要。 </li></ol></li></ol> 3.筛选的文化整合子2浓度为2.5mM。如果需要的话，洛伦茨（2011）24概述了在这个杂志的早期发行的PCR优化和故障排除方法。 <ol><li>煮DNA制备： <ol><li>分配100μl的富集培养物进入无菌0.5毫升PCR管。加热样品至99℃下进行10分钟，用水浴或加热块。卡扣冰上冷却2分钟。 </li><li>微量在14000×g离心5分钟以沉淀细胞碎片。回到冰。 DNA样品可以在-20℃冷冻直至需要的，但必须在冰上解冻。 </li></ol></li><li>筛选使用PCR 1类整合： 注：从O / N混合培养培养筛选使用HS463a / HS464 PCR协议（ 表1）1类整合。 <ol><li>解冻所有的PCR试剂在冰上，远离任何潜在SOU污染的DNA的RCE中。弥补48微升的PCR mastermix每个样品，包括阴性对照（ 表2）。免除48微升PCR主混合物中，每管使用障碍的提示。 </li><li>加入2微升的DNA解冻使用障碍的提示各PCR管。在PCR热循环仪运行相应的程序。 </li><li>为了评估扩增的结果，electrophorese 7微升PCR产物在2％琼脂糖凝胶倾和在TBE缓冲液（90毫摩尔Tris-硼酸，2mM EDTA）中运行。包括分子量标记，如一个100bp的阶梯。 </li><li>交的染色用DNA染色的凝胶，和可视化使用UV透照。检查是否有已在阴性对照无扩增。在大约470基点的任何特定样品强大的乐队表明混合培养包含携带1类整合细菌。纯粹的殖民地现在从返回的阳性PCR任何混合培养物中分离。 </li></ol></li></ol><ol><li>连续稀释和扩散板： 注意：为了分离单菌落从PCR阳性混合培养物中，混合培养物连续稀释10倍的磷酸钠缓冲液中，然后镀以回收单菌落。 <ol><li>加入1毫升混合培养至9毫升0.1M磷酸钠缓冲液，pH 7.0和通过倒置混合。加入1毫升的稀释液，以进一步9毫升磷酸盐缓冲液中，并重复直到10 -8系列稀释为止。 </li><li>蔓延100微升10 -4至10 -8稀释到LB琼脂，一式两份。孵育板O / N在用于初始混合培养相同的温度。板然后可被储存在4℃直到需要，但是最好是处理单菌落相当迅速。 </li></ol></li><li>单个菌落选择和煮DNA制备： <ol><li>从串行稀释板，本身挑单个菌落lecting许多不同菌落类型成为可能，使用的标准，如集落的大小，形状和颜色。使用无菌牙签选择从传播板单菌落。 </li><li>触摸牙签的菌落，然后转移到含有100微升无菌水PCR管。如果很大数量的菌落被筛选，然后菌落可以在微量滴定盘制备。旋转的手指之间的牙签撞出一些细胞进入水中。 </li><li>使用相同的牙签，接种LB平板与殖民地的连胜。大量菌落可以存储在标记板如果每个条纹为约1厘米长。直到选择分离适当数量的重复步骤1〜3。 </li><li>孵育LB平板O / N，在相同的温度与用于初始浓缩培养。培养物然后可被储存在4℃下直到需要。 </li><li>对于从菌悬液准备DNA，按照步骤为OUtlined 3.1节。对于纯培养裂解物的筛选，执行HS463a / HS464 PCR在3.2中列出。隔离返回一个正的PCR将用于制备基因组DNA（部分5），以及用于进一步的分析。 </li></ol></li></ol> 5.基因组DNA提取整合子阳性单菌落使用珠击25 <ol><li>接种5毫升LB肉汤与纯培养物中分离的第4.2节的样品。孵育O / N，振荡被用于初始富集培养的相同的温度。 </li><li>沉淀细胞在4000 XG 7分钟，然后取出上清。悬浮在一个裂解矩阵E快速制备管1毫升CLS-TC解决方案沉淀的细胞。使用珠打浆机，处理该样本以5.5米/秒，30秒。 </li><li>在14000×g离心5分钟离心管中并回收700微升上清液进入无菌1.5mL管中。 </li><li>加入700μl稀释1结合矩阵：5用6M关idinium硫氰酸并混合在RT 5分钟。离心机在14000 xg离心1分钟，弃去上清液。 </li><li>添加800微升盐乙醇洗涤（70％乙醇，0.1M乙酸钠）和涡流直至沉淀完全重悬。在14000×g离心洗5分钟，室温，离心1分钟，弃上清。确保所有上清液已被去除，使用微量如果必要的话，允许风干5分钟。 </li><li>通过上下吹打悬浮于200μl的TE中（10mM的Tris-HCl，1mM的EDTA，pH7.6）中的小球基质。在室温下孵育3分钟。离心机在14000×g离心3分钟，并转移160微升洗脱的DNA的到无菌管中。运行在2％w / v琼脂糖凝胶的基因组DNA提取的等分试样来检查DNA提取的得率和完整性。将纯化的基因组DNA被储存在-20℃，并需要用于PCR检测时解冻在冰上。 </li></ol> 6.诊断的PCR和DNA测序注意：在第5制备的基因组DNA将被用于所有的诊断的PCR，并以确认阳性测试为1类整合。 <ol><li>冰上解冻基因组DNA，短暂涡旋的DNA样本和脉冲旋在14000 XG为20秒。重复的PCR用HS463a / HS464（ 表1），确认该菌株是积极为471 bp的扩增（3.2节）1类整合子整合酶基因。 </li><li>阳性培养物，以种的水平鉴定是由小亚基rRNA基因（16S rDNA的）的分析来实现的。使用引物F27 / r1492（ 表1）扩增16S基因。检查采用电泳PCR产物。所有的细菌指标应生成16S扩增大约1450碱基对。 </li><li>序列的16S rRNA基因的扩增子，以确定物种的身份，但由于有可能是多个正的PCR从步骤6.2，并且许多这些阳性的将是相同的细菌种类，我们将首先区分不同物种全光照在16S PCR产物克酶切。 <ol><li>消化16S的PCR的等分试样用限制酶的Hinf 1。任何酶识别4 bp的切割位点会做，但1 的Hinf从生成目标16S良好的多样性，是一个可靠的，廉价的酶。 </li></ol></li><li>成立了30微升限制性消化mastermix（ 表2）在无菌试管，加入20微升未purfied 16S PCR产物。孵育在37℃水浴O / N。检查消化在2％琼脂糖凝胶。 </li><li>分数消化图案的Hinf所产生的1。隔离用相同的16S限制信息有可能是相同的物种。而不是每次测序16S PCR产物，序列的Hinf每1限制模式的最三名代表。 </li><li>纯化使用商业试剂盒的16S PCR产物。单链核酸外切酶试剂盒可用于，但基于列的或沉淀的方法也工作良好以除去unincorporated引物和单链DNA。序列使用引物R910（ 表1），所指定的测序设施建立反应16S扩增子。 </li><li>使用产生的DNA序列与功能BLASTN（询问NCBI数据库的DNA <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ）。大于97％核苷酸同一性通常取为物种身份。 </li></ol> 7.映射和整合子和磁带阵列的表征注：1类整合来自人类主宰的生态系统所产生很可能是所有样品中最常见的整合。这些整合都具有最新单曲的起源，因此具有高度保守的DNA序列5。 <ol><li>一类区分1整合来自人力资源和所产生的那些自然发生环境样品中通过执行PCR引物与设置intI1F165 / intI1R476 10（ 表1）。 注：1级临床或共生菌整合将产生约300 bp的产品，而环保类1整合不会产生任何产品。 </li><li>通过放大盒阵列引物HS458 / HS459表征临床整合。该PCR将扩增intI1和3&#39;保守区段之间的区域中至多暗盒阵列的末端。因为盒的数目和身份是可变的，所述PCR产物的大小也将有所不同。 </li><li>纯化的扩增子和测​​序使用每个的扩增引物的DNA。使用序列询问使用BLASTN功能（DNA数据库<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> ）。 </li><li>确保盒的命名遵循标准化术语9，因为许多数据的证词整合基因盒■使用不一致或不明确的名称。特别注意的新基因盒，因为这些可以编码新的表型，包括基因，赋予提高传播能力，致病性或毒性8,21。 注：临床前的1类整合形式仍然散发自然的环境。特别是，那些与活性Tn的402转座子最感兴趣26。这些整合的盒的阵列将不会与引物组HS458 / HS459放大，但可以与MRG284 / MRG285引物组（ 见表1）被放大，产生可变尺寸依赖于盒式内容的扩增子。 PCR产物应纯化和DNA测序为7.2概述。 </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples.">Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples.</a> Env. Microbiol. 1096, 17-23 (2014).</li><li>Sajjad, A., Holley, M. P., Labbate, M., Stokes, H., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module.">Preclinical class 1 integron with a complete Tn402-like transposition module.</a> Appl. Env. Microbiol. 77 (1), 335-337 (2011).</li><li>Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. 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=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

作者必须披露相关一无所获。

整合及其相关基因盒的识别可能是在预测的新机会病原体的出现，跟踪途径病原体进入人类食物链，并确定新的抗性的关键步骤和毒力决定8,21,26。本文的目的是描述筛选样品1类整合，其特征阵列盒，并确定其所在的细菌种类精简的办法。在协议中的关键步骤涉及微生物好做法，并防止污染的PCR将产生误报。 这里所描述的协议可以很容易地修改，以检测其他临床相关?...

抗生素的发现是20 世纪最伟大的科学成就之一。然而，抗生素的使用和滥用，导致对抗生素有抗药性的细菌的快速发展，而这些现在在构成21 世纪严重威胁到公众健康。细菌耐药株大多数治疗方案的兴起，引起了我们正在进入一个时代，抗菌药物不再有效1,2的可能性。 赋予抗生素抗性的遗传机制是一个古老的制度，由百万年3早于人类和抗生素的选择压力。移动遗传元件，如质粒，转座子，基因组岛，一体化接合元件和整合子可以传播抗生素抗性基因（ARG）内和细菌物种4之间。其中，整合都起到ARG传播的核心作用，尽管事实上，他们依靠质粒和转座子动员和插入到细菌基因组中5。整合捕获使用整合子整合酶基因盒，然后表达使用整合编码启动子6,7-（ 图1）盒。 整合基因盒是由单个开放阅读框（ORF），其产品能够赋予对抗生素或消毒剂8的小型移动元件。 1类整合是从临床分离5，在那里他们已经集体过130个不同的抗生素耐药基因盒9收购最常用的回收整合。 1类整合传播到人类的相关共生菌和致病菌产生含有大量的这些遗传元件10的人类废物流。据估计，10 19的细菌包含1类整合是通过污水污泥每年我发布n中的英国11。因此，毫不奇怪，1类整合赋予抗生素抗性现在正在野生鸟类，鱼类和其他野生动物12-14菌群检测。整合释放回环境构成了显著的公共健康威胁，因为收购新基因盒和复杂的重组与其他移动元素仍然存在，特别是在污水处理厂和其他水体15-18。自然环境就变成一个招兵的新的阻力因素和条件致病菌19,20。新型含整合细菌和新的ARG可以绕回到人类社会通过被污染的水和食物21,22。 ARG游戏环境监测是理解和未来23管理抗生素耐药性的关键战略。特别是，要注意食品要被生吃或轻熟，因为这些本作的新要素流动和​​病原体传播的最大威胁。 在这个协议中，用于检测，鉴定和表征1类整合并在食品及其相关基因盒一个简化的方法概述（ 图2）。使用的培养和聚合酶链反应（PCR）的组合，整合可迅速在复杂细菌群落和个体分离物检测。方法来识别细菌和整合相关基因盒的构象和身份的物种中给出。该方法适用于范围广泛的植物和动物的食物和典型的工作流程的例子给出了每个这些食物类型。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>GoTaq Colourless Mastermix</td>
			<td>Promega</td>
			<td>M7132</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>RNAse (Ribonuclease A from bovine pancreas)</td>
			<td>Sigma</td>
			<td>R6513-10MG</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>HinFI restriction enzyme</td>
			<td>Promega</td>
			<td>R6201</td>
			<td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>GE Healthcare</td>
			<td>27400701</td>
			<td>Used as a size standard on all agarose gels</td>
		</tr>
		<tr>
			<td>GelRed DNA stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate</td>
			<td>Life Technologies</td>
			<td>AM9422</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>CLS-TC Solution</td>
			<td>MP Biomedicals</td>
			<td>6540409</td>
			<td>Resuspension solution used at the begining of the genomic DNA extraction</td>
		</tr>
		<tr>
			<td>Lysing Matrix E FastPrep tubes</td>
			<td>MP Biomedicals</td>
			<td>116914500</td>
			<td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td>
		</tr>
		<tr>
			<td>binding matrix</td>
			<td>MP Biomedicals</td>
			<td>116540408</td>
			<td>Diluted 1:5 with 6M guanidinium thiocyanate and used in the genomic DNA extraction method.</td>
		</tr>
		<tr>
			<td>Fast Prep machine</td>
			<td>MP Biomedicals</td>
			<td>Number of options available</td>
			<td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information</td>
		</tr>
	</tbody>
</table>

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.

混合培养物和细菌分离的intI1筛选引物组HS463a / HS464 PCR可以用来检测类整合-整合酶基因，intI1（ 图1）的存在。这个引物组可以很好地用于在混合培养物检测intI1，并且也用于筛选从扩散板（ 图2）收获的细菌菌落。阳性分离应在471碱基对使用该引物组（ 图3A）产生单一强条带。大多数阳性菌株将携带的，源自人类或他们的农业?...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and 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 ...

screening-foodstuffs-for-class-1-integrons-and-gene-cassettes

Research

JoVE Journal

Environment

8.9K 次观看.  Macquarie University. This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs. 

视频: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

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 ...

科研

环境科学

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

半高通量筛选潜在的抗干旱的生菜（<em&gt;莴苣</em&gt;）种质资源

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

总木质素，木质素单体，和酶促糖推出：顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

测定掠夺取食行为<em&gt; Pristionchus</em&gt;及其他线虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

在用水筛查内分泌活动市售的<em&gt;体外</em&gt;式激活生物测定

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

提取和表面活性剂的特性，从大气气溶胶

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

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 ...