Name: a whole cell bioreporter approach to assess transport and bioavailability of organic contaminants in water unsaturated systems
Uploaded: 2014-12-24T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems at JoVE.com

Funding by the German Environmental Foundation (DBU) is acknowledged. The authors thank Ute Kuhlicke for technical help with CLSM analysis and Birgit W&#252;rz, Rita Remer, and Jana Reichenbach for skilled experimental help. The authors would particularly like to thank Prof. Jan Roelof van der Meer and Dr. Robin Tecon for fruitful discussion and providing the bioreporter strain. It contributes to the &#8216;Chemicals in the Environment&#8217; (CITE) research program of the Helmholtz Association.

1.准备菜肴，幻灯片和孵化商会<ol><li>准备以下材料每缩影:一个大的塑料培养皿底部（D = 10厘米），一个被修改的（见步骤1.2）的小塑料培养皿底部（D = 5厘米）与盖和三腔一个计数室幻灯片。 </li><li>取的培养皿底部零件所需的数字（D ​​= 5厘米）。用锯恰好适合幻灯片（26毫米边长）拆下边缘的一部分。消毒系统，浸泡培养皿底部和盖在70％乙醇的O / N和干燥它们至少2小时在流动柜在UV光下。存储培养皿在一个密封的塑料容器，这也被暴露在UV光2小时。 </li><li>擦滑动用70％乙醇和所需数量的空气干燥。包裹滑动铝箔，并把它们在马弗炉中进行5小时，在450℃以除去可能的污染物。 </LI&gt; <li>准备玻璃孵化室带盖。清洗，用70％的乙醇和暴露室开放于UV光至少2小时在流动柜。 注:大干燥器（直径约30厘米），用盖子可以用于这一目的;每个干燥器可容纳多达10缩影设置。 </li></ol> 2.准备媒体与文化<ol><li>运行20平行缩影，得到百毫升改性胰蛋白胨-酵母培养基（MTY）14，约150毫升的21℃的基本培养基16（MM），将5ml基本培养基琼脂甲酯（MMA; 0.5％，重量/体积），三马铃薯葡萄糖琼脂平板上（PDA; 1.5％，重量/体积）为P.霉 ，4空白板，其中之一PDA平板含有2mg L -1酮，四琼脂糖平板（3％，重量/体积）含0.3克·毫升-1活性炭和40mg感在2毫克的部分。 </li><li>制备含50毫克卡那霉素的L -1 MTY（卡那霉素是requirED保持EGFP报告质粒）和MM含10毫米醋酸细菌培养。使用MM的无乙酸以制备0.5％的MMA为细菌在微观上固定和倾1.5％的PDA平板（各20毫升）中进行栽培的终极腐霉和缩影设置。 </li><li>接种P.一些菌丝霉三个新鲜PDA平板孵育在黑暗中在25℃下进行约72小时。然后确保这些板块都长满完全新鲜和柔软的菌丝体。 </li><li> B.启动两种文化sartisoli RP037-MCHE在50ml MTY的从甘油贮存。孵育在30℃，230转旋转烧瓶运动O / N。 </li><li>测量培养的光密度（OD）在578纳米和离心机在1000×g离心10分钟（为B. sartisoli RP037-MCHE，预期的OD 578为约1.0和细胞处于指数期）。弃上清液并重新悬浮细胞在MM中的一个适当的量来达到的OD 0.4 578。接种20毫升的MM含有50mg L-1用400微升重新悬浮的细胞的卡那霉素。孵育在30℃，230转旋转烧瓶运动O / N。 </li><li>衡量MM文化的OD 578（为B. sartisoli RP037-MCHE，预计OD 578大约是0.2和细胞在早期指数期）。离心培养在2000×g离心10分钟，并重新悬浮细胞中的MM适量以达到0.4的OD。混合50微升用500μl温暖，液体的MMA的细胞悬浮在2ml管中并储存在50℃，直到使用。用150微升的细胞中的MMA的每个缩影。 </li><li>倾含有活性碳的琼脂糖凝胶片。融化琼脂糖（3％;重量/体积）在双蒸水。用活性炭调匀（0.3克毫升-1），将混合物倒入塑料培养皿（D = 10厘米）。 </li></ol> 3.缩影安装注意:完整的缩影设置在图1中示意性地描绘的，请参阅图1为所有以下各个步骤。下面的步骤被描述一个样品（SAM）与菌丝传输载体和三种不同的控制设置（CON AIR中 ，CON NEG，CON POS）的制备。所有不同的设置的摘要可以在表1中可以找到。 <ol><li>取的培养皿和幻灯片期望数量和它们下一个流柜暴露于UV光30分钟。 </li><li>放置小培养皿内的大的，并通过小培养皿的间隙配合的幻灯片。取暖细胞悬浮液中的MMA的一个样本，并添加150微升到中间的一个滑动的空腔。直到所有幻灯片的中间空腔重复此步骤，充满了MMA。等待5分钟的MMA凝固。 </li><li>使用1厘米的打孔器冲出来的圆形贴片从空白PDA板。放置一个贴剂以2mm旁边的MMA的小皮氏培养皿内的距离。添加该促进在设置菌丝生长（PDA 3中图1）。 </li><li>切弯PDA补丁的机械屏障。使用干净和无菌小培养皿底部，并将其按入一个PDA板。用刮刀切另一个较小环以0.5厘米的距离成其导致一个PDA的环琼脂。 <ol><li>切出一块，这恰好装配到小培养皿中的缩影设置的间隙，并将其放置在滑动的顶部的间隙内。确保MMA和圆形的PDA屏障之间的距离为约2毫米。关闭小培养皿轻轻按压入在PDA屏障的盖子。 </li><li>重复步骤3.4和3.4.1与含有2g L的PDA板-1对放线菌酮。 注意:这是控制设置为空中运输（CON AIR）对bioreporter细胞，由于菌丝体是由放线菌酮抑制，不长满MMA。 </li></ol></li><li>使用1厘米的打孔器冲出来的是空白PDA板的圆形贴片。使用另一个0.5厘米打孔器切出第一补丁的中间。放置所产生的小的PDA型环以1mm旁边的PDA屏障（PDA 1中图1）的距离。 </li><li>添加2毫克流感进入PDA 1.用孔1厘米的打孔器切出从PDA板长满P.圆形补丁霉和放置一个补丁（PDA 2在图1中）底部向下到PDA的环，使得菌丝垫朝向FLU晶体。 <ol><li>无需重复添加FLU晶体上一步。 注意:这是阴性对照设置（CON NEG）来确定背景荧光bioreporter细胞。 </li></ol></li><li>使用1厘米的打孔器冲出来的圆形贴片含有活性炭琼脂。将4个贴片在每一个缩影设置，以进一步降低气体浓度FLU（ 图1）。 </li><li>准备阳性对照（CON POS）。一些FLU晶体添加到200微升的MMA细胞悬液，并把它放在一个空的幻灯片的一个腔。 </li><li>放置一个培养皿（D = 10厘米），用活性炭对孵育室的底部的一些粉末，以尽量减少在腔室中的气态FLU浓度。此外，放置在底部四到五50毫升烧杯用无菌水以保持高湿度的室中。 <ol><li>转移缩影设置成孵化室和孵化在25℃96小时。将样品随机在不同的生长室，以排除位置的影响。 注:此孵化时间AP层数为卵菌P.霉 。它可能是用于其它生物体不同。 </li></ol></li></ol> 4.激光共聚焦显微镜<ol><li>理想的情况是使用激光共聚焦显微镜的设置与正置显微镜。 注意:它可以配备有常规的激光器提供例如用于激励或具有可调白光激光源488，561和633纳米的线条。 </li><li>使用以下设置收集的图像:激发:490纳米（EGFP）和585纳米（mCherry）在适当的激光强度（同时激励）;发射范围:500〜550纳米（EGFP）和605至650纳米（mCherry）;物镜:63X 0.9 NA水浸入（由于其工作距离长）;步长大小对于z轴堆栈:0.5微米。 </li><li>开放缩影设置和使用手术刀去除PDA 1，2，3和PDA的屏障。放置在MMA的顶部玻璃盖玻片并按下它轻轻地除去气泡。在显微镜阶段安装幻灯片。加的水的液滴在th之上Ë盖玻片并用mCherry设置找bioreporter细胞样品中。 </li><li>获取样品的快速浏览和使用电热环比下查找表的红色（mCherry）和绿色（EGFP）信道优化信噪比。选择在样本的随机位置来分析bioreporter荧光。 注:菌丝可能显示自发荧光在bioreporter发射（ 例如，绿色自发荧光）17的范围内。一定要选择无菌丝地区，如果是这样的话。 </li><li>定义和在绿色（EGFP）和红色（mCherry）信道的各位置记录的z栈。通过长时间曝光，以萤光光或激光避免样品漂白。 <ol><li>对10个随机，均匀的分布位置，重复前面的步骤。 </li></ol></li><li>重复使用相同的设置进行测试轨道（SAM），CON AIR，CON NEG和CON POS的所有样品的所有步骤。 </li></oL&gt; 5.图像分析注意:要分析红色（mCherry）和绿色（EGFP）的荧光在记录的z栈，其中包括选择的自由软件ImageJ的18（http://rsb.info.nih.gov/ij/download.html）可以是使用。 <ol><li>请确保安装logi_tool插件（http://downloads.openmicroscopy.org/bio-formats/4.4.10/）。 </li><li>在ImageJ的打开文件。选择"拆分通道"并关闭绿色通道。用宏"mCherry区"作为补充代码文件到Z堆栈量化红色荧光区。在宏观调整首选设置（以粗体显示）。 <ol><li>输出与所测量的像素在阈值以上（以上所定义的最小尺寸）的Z-堆栈的表。 注:所有值的总和是一个红色像素（区域）中的叠层的总数。 </li></ol></li><li>在ImageJ的打开文件。选择"拆分通道"，并接近红色通道。使用宏<html> 216;绿色荧光蛋白INT"作为补充代码文件，以量化的绿色荧光强度Z-堆栈。在宏观调整首选设置（以粗体显示）。 <ol><li>输出与平均强度在Z堆栈的所有绿色物体高于阈值（及以上定义的最小尺寸）和区域的表格。为了计算在堆栈绿色像素的总强度，乘以各意味着由相应的区域的强度。 </li></ol></li><li>计算相对的eGFP感应（eGFP的REL）: <img alt="式（1）" src="/files/ftp_upload/52334/52334eq1.jpg" width="300px" /> （1） 注意:由于mCherry表达组成型，eGFP的相对的结果值是的eGFP的一定量的细胞的表达量的相对量度。也请参考表3的绿色荧光蛋白相对计算的进一步信息。 </li></ol>TLE"&gt; 6。化学定量注:微观设置也可被用于执行的易位FLU量有或没有非生物污染物水槽化学定量。 <ol><li>包装15毫升玻璃小瓶，1毫升GC / MS小瓶和插入铝箔。填充用Na 2 SO 4蒸发器皿;将所有在马弗炉中进行5小时，在450℃以除去可能的污染物。存储活化干燥器的用Na 2 SO 4内。 </li><li>切割PDMS涂覆的玻璃纤维成0.5厘米长。通过摇动四倍2小时的新鲜甲醇洗纤维。重复洗涤步骤再四次用超纯水和存储清洁纤维在超纯水中，在密封的玻璃小瓶中。 </li><li>制备1.5升的甲苯/丙酮（3:1，V / V）含有10μgL -1的菲-D 10。 </li><li>执行步骤1，2和3的协议。准备微观无bioreporter细胞来研究FLU运输（SAM（ - ）和CON AIR（ - ））中，用bioreporter细胞（SAM）来验证FLU降解。 <ol><li> 可选（无bioreporter细胞）:将镀膜玻璃纤维3 PDMS（通常用于SPME实验）作为内SAM的MMA人工和可量化的污染物接收器（ - ）用细镊子。 </li><li>删除使用细镊子玻璃纤维和每一个与插入转移到GC / MS小瓶。 </li><li>加入200μl甲苯和垂直摇O / N，并通过GC / MS分析。使用表2中提供的设置。 </li></ol></li><li>从中间空腔转移的MMA入15ml玻璃小瓶中，并加入约用Na 2 SO 4 3铲。调匀用抹刀和加入10毫升溶剂。旋涡的混合物，并在黑暗中晃动水平O / N。 <ol><li>转移1001，L样品放入GC / MS小瓶插入。添加10微升苊-D 08（10毫克L-1），并使用如在6.4.3中描述的相同的程序，通过GC / MS法进行分析。 </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+8+Concepts+in+Mycorrhizal+Research.">Ch. 8 Concepts in Mycorrhizal Research.</a> Handbook of Vegetation Science. Vol. 19, 195-222 (1996).</li><li>Furuno, S., 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=Fungal+mycelia+allow+chemotactic+dispersal+of+polycyclic+aromatic+hydrocarbon-degrading+bacteria+in+water-unsaturated+systems.">Fungal mycelia allow chemotactic dispersal of polycyclic aromatic hydrocarbon-degrading bacteria in water-unsaturated systems.</a> Environ. Microbiol. 12, 1391-1398 (2010).</li></ol>

The authors declare that they have no competing financial interest.

所提出的缩影设置证明适合学习的空间分离的化学品的生物利用度，以降低摄取和运输的菌丝体后的生物。防止部分挥发性化合物的潜在的气相输送与细菌bioreporter细胞可以可视化，而不精心样品制备并因此与敏感的系统的干扰最小。同时，样品的化学分析可以容易地进行，允许有一个良好的控制获得的结果的和的总运输定量。然而，一些点必须仔细之前和在执行实验审议。很重要的一点就是保?...

人口稠密由多种微生物1,2的土壤如细菌。然而，在此栖息条件具有挑战性，特别是在水的供应3项。菌永久需要寻找在异构环境4的最佳条件，但没有连续水膜是导致受限可移动性5妨碍他们自由传播。此外，溶质的扩散率（ 例如，营养素）的不饱和的条件6下降低。因此，细菌和营养物往往物理上分离和营养辅助限于3。其结果是，传输载体的化学化合物，它不需要连续水相可有助于克服这些限制。事实上，很多微生物如真菌和卵菌已经开发出一种丝状增长方式，使他们通过空气填充孔隙成长从而达到和MOBIlizing也物理长距离分离营养素7和8含碳物质。他们甚至可以作为其提供糖和其他能源，以细菌9生物的运输载体。摄取和运输中菌丝生物体也已显示对疏水性有机污染物如多环芳烃（PAH）在终极腐霉 10或在丛枝菌根真菌11。由于PAH是无处不在，难溶于水的污染物在土壤中12，菌丝体介导的运输可能有助于提高生物利用度污染的潜在细菌降解。装置10而污染物输送的总量，可以通过化学直接定量，污染物通过菌丝体降解细菌和其它生物体输送的生物利用度不能容易地进行评估。 以下协议呈现给评估myce的影响的方法LIA对污染物的生物利用度要以直接的方式细菌降解;它允许收集有关污染物的微生物生态系统的时空影响的信息。我们描述了如何建立一个复杂的不饱和缩影系统通过将一个物理上分开PAH点源通过菌丝体的运输载体的PAH降解菌bioreporter模仿气水界面土壤。因为空中交通被排除，菌丝基于交通工具上的PAH的生物利用度对于细菌的效果，可以研究在一个孤立的方式。更详细地说，三环多环芳烃芴，菌丝生物体终极腐霉和细菌bioreporter 伯克霍尔德sartisoli RP037-MCHE 13中所描述的缩影设置施加。细菌B.最初构建sartisoli RP037-MCHE研究菲通量到电池14，并表示增强型绿色荧光蛋白（EGFP）的PAH通量的结果细胞中，而红色荧光mCherry表示组成。详细信息，记者建造由TECON 等 13在初步测试中给出，细菌没有透露游泳，只有非常缓慢蜂拥能力。它能够在作为致密悬浮在菌丝顶端施加缓慢迁移对终极腐霉的菌丝。因为细菌嵌入琼脂糖在以下方案，上菌丝迁移并没有出现。 使用共聚焦激光扫描显微镜（CLSM）中，bioreporter细菌，可以直接在微观可视和EGFP的表达可以相对于被量化到细胞的量（正比于mCherry信号）与该软件ImageJ的的帮助。这允许定性在不同的情景进行比较生物利用度（ 即，更高或更低）。 FLU被发现是后由P.菌丝运输生物利用霉 （ 即，它较阴性对照更高）。此外，该协议描述了如何量化通过化学手段菌丝体介导的转运的总量，并使用硅被覆的玻璃纤维（固相微萃取纤维）中相同的缩影验证污染物的生物利用度。使用此设置一个缩影成果已发表 ​​和体育相结合的探讨霉 ，芴和B. sartisoli RP037-MCHE 15。在这里，重点在于一个详细的方法说明和协议的潜在缺陷的鉴定潜在的进一步应用提供了这方面的知识。进一步的应用可涉及各种真菌，细菌种类（ 例如，从污染的场所），以及其它污染物（ 例如，农药）或杂质的供应（ 例如，年龄土壤）。

<table><tbody><tr><td>Confocal Microscope</td><td>Leica</td><td></td><td>TCS SP5X, LAS AF - Version 2.6.1; or equivalent CLSM</td></tr><tr><td>GC HP 7890 Series GC and Agilent 5975C MSD</td><td>Agilent</td><td></td><td>an equivalent GC/MS may be used</td></tr><tr><td>GC capillary column J&amp;amp;W 121-5522&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Agilent</td><td></td><td></td></tr><tr><td>Cork borer</td><td>Fisher Scientific</td><td>12863952</td><td>or any other</td></tr><tr><td>Cover slips</td><td>Marienfeld</td><td>107222</td><td>High performance, No.1.5H</td></tr><tr><td>GC/MS insterts</td><td>WICOM</td><td>WIC 47080</td><td></td></tr><tr><td>GC/MS vials 2 ml</td><td>WICOM</td><td>WIC 41150</td><td></td></tr><tr><td>Lids / septa for screw cap vials</td><td>DIONEX</td><td>49463 / 049464&amp;nbsp;</td><td></td></tr><tr><td>Lids for GC/MS vials</td><td>WICOM</td><td>WIC 43948/B</td><td></td></tr><tr><td>Objective Slides</td><td>Menzel</td><td></td><td>ordinary</td></tr><tr><td>PDMS coated glass fibers</td><td>Polymicro Technologies, Inc.</td><td></td><td>V (PDMS) = 13.55&amp;nbsp;&amp;plusmn;&amp;nbsp;0.02&amp;nbsp;&amp;micro;l&amp;nbsp;m&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Petri Dishes small / big</td><td>Greiner</td><td>633-102 / 628-102</td><td></td></tr><tr><td>Screw cap vials 40 ml</td><td>DIONEX</td><td>48783</td><td>other glass vials may be used</td></tr><tr><td>Screw cap vials 60 ml</td><td>DIONEX</td><td>48784</td><td>other glass vials may be used</td></tr><tr><td>Acenaphthylene d&lt;sub&gt;08&lt;/sub&gt;</td><td>Dr. Ehrenstorfer</td><td>C 20510100</td><td></td></tr><tr><td>Acetone</td><td>Carl Roth</td><td>9372.2</td><td></td></tr><tr><td>Activated carbon</td><td>Sigma-Aldrich</td><td>242276-1kg</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth</td><td>2267.4</td><td></td></tr><tr><td>Fluorene</td><td>Fluka</td><td>46880</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Carl Roth</td><td>T832.2</td><td>50 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>P7171</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Minimal Medium:&lt;/strong&gt; 100 ml &lt;strong&gt;Solution 1&lt;/strong&gt; + 25 ml &lt;strong&gt;Solution 2&lt;/strong&gt; + 5 ml &lt;strong&gt;Solution 3&lt;/strong&gt; ad. 1,000 ml aqua dest</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 1&lt;/strong&gt;</td></tr><tr><td>Ammonium sulfate</td><td>Carl Roth</td><td>3746.1</td><td>5 g L&lt;sup&gt;-1&amp;nbsp;&lt;/sup&gt;</td></tr><tr><td>Magnesium chloride x 6&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>2189.1</td><td>1 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Calcium nitrate x 4&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>P740.1</td><td>0.5 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 2&lt;/strong&gt;</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth</td><td>P030.1</td><td>55.83 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Monopotassium phosphate</td><td>Carl Roth</td><td>3904.1</td><td>20 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Solution 3&lt;/strong&gt; (pH 6.0)</td></tr><tr><td>Disodium EDTA</td><td>MERCK</td><td>1084180250</td><td>0.8 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Iron(II) chloride x 4&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>MERCK</td><td>1038610250</td><td>0.3 g L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Cobalt(II) chloride x 6&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>T889.3</td><td>4 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Manganese(II) chloride x 1&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>4320.2</td><td>10 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Copper(II) sulfate</td><td>Carl Roth</td><td>P023.1</td><td>1 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Sodium molybdate x 2&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>0274.1</td><td>3 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Zinc chloride</td><td>MERCK</td><td>1088160250</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Lithium chloride</td><td>Carl Roth</td><td>P007.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Tin(II) chloride x 2&amp;nbsp;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>4473.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Boric acid</td><td>Riedel-de-Haen&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>11606</td><td>1 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Potassium bromide</td><td>Carl Roth</td><td>A137.1</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Potassium iodide</td><td>Carl Roth</td><td>6750.1</td><td>2 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Barium chloride</td><td>Carl Roth</td><td>4453.1</td><td>0.5 mg L&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>MMA</td><td></td><td></td><td>Minimal medium + agarose 0.2%</td></tr><tr><td>Phenanthrene d&lt;sub&gt;10&lt;/sub&gt;</td><td>Dr. Ehrenstorfer</td><td>C 20920100</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Potato Dextrose Agar:&lt;/strong&gt; 24 g L&lt;sup&gt;-1&lt;/sup&gt; broth + bacto-agar 1.5%; pH 6.8</td></tr><tr><td>Potato Dextrose broth</td><td>Difco/ Beckton Dickinson</td><td>254920</td><td></td></tr><tr><td>Bacto-agar</td><td>Difco/ Beckton Dickinson</td><td>214040</td><td></td></tr><tr><td>Sodium acetate x 3 hydr.</td><td>Carl Roth</td><td>6779.1</td><td></td></tr><tr><td>Sodium sulfate</td><td>MERCK</td><td>1066495000</td><td></td></tr><tr><td>Toluene</td><td>MERCK</td><td>1083252500</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;mTY medium:&lt;/strong&gt; 3 g&amp;nbsp;L&lt;sup&gt;-1&lt;/sup&gt; yeast extract, 5&amp;nbsp;g&amp;nbsp;L&lt;sup&gt;-1&lt;/sup&gt; bacto tryptone and 50 mM NaCl</td></tr><tr><td>Yeast extract</td><td>Merck</td><td>1037530500</td><td></td></tr><tr><td>Tryptone</td><td>Serva</td><td>4864702</td><td></td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>ImageJ with logi tool plugin</td><td></td><td></td><td>http://rsb.info.nih.gov/ij/download.html and http://downloads.openmicroscopy.org/bio-formats/4.4.10</td></tr><tr><td>&lt;em&gt;Pythium ultimum&lt;/em&gt; strain 67-1</td><td></td><td></td><td>Obtained from the lab of Dr. Christoph Keel; Department of Fundamental Microbiology, University of Lausanne, Switzerland</td></tr><tr><td>&lt;em&gt;Burkholderia sartisoli&lt;/em&gt; RP037-mChe</td><td></td><td></td><td>Obtained from the lab of Prof. Jan Roelof van der Meer; Department of Fundamental Microbiology, University of Lausanne, Switzerland</td></tr></tbody></table>

a whole cell bioreporter approach to assess transport and bioavailability of organic contaminants in water unsaturated systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

这里介绍的结果已经较早公布的15。请参考文章进行详细的机械和环境的讨论。 通过CLSM图像记录后，最大强度投影可使用相应的显微镜软件或ImageJ的获得样品的一个第一视觉印象和对照（ 图2）进行。以后，该数据集可以以显示由特定的可视化软件有意义特征被投影不同。阳性对照（CON POS）显示明显的绿色荧光蛋白的诱导（ 图...

Watch this Scientific Journal Video about A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems at JoVE.com

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

与鼻疽sartisoli RP037-MCHE全细胞bioreporter法的开发是为了检测有机污染物（ 即芴）供细菌降解菌丝桥空气填充孔隙中水不饱和模型系统主动转运后的分数。

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

a-whole-cell-bioreporter-approach-to-assess-transport-bioavailability

Research

JoVE Journal

Environment

10.3K 次观看.  Helmholtz Centre for Environmental Research - UFZ. 与鼻疽sartisoli RP037-MCHE全细胞bioreporter法的开发是为了检测有机污染物（ 即芴）供细菌降解菌丝桥空气填充孔隙中水不饱和模型系统主动转运后的分数。 

视频: A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

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 ...

科研

环境科学

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

通过活生物膜表征电子传输

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

化学

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

生物工程

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

细胞的细胞大分子运输检测在植物利用基因枪法

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

生物学

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

摘要

探索更多视频

此视频中的章节

在用水筛查内分泌活动市售的

流式细胞仪对水生生物膜的表征

通过活生物膜表征电子传输

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

方法表征的共同发展生物膜和生境异质性

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

细胞的细胞大分子运输检测在植物利用基因枪法

通过电穿孔内在和生物分子荧光观察生活中的微生物

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

摘要

探索更多视频

此视频中的章节

在用水筛查内分泌活动市售的

流式细胞仪对水生生物膜的表征

通过活生物膜表征电子传输

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

方法表征的共同发展生物膜和生境异质性

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

细胞的细胞大分子运输检测在植物利用基因枪法

通过电穿孔内在和生物分子荧光观察生活中的微生物

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统