We sincerely thank Thomas Howe and J&#246;rg Carsten for the programming and technical support with the liquid handling systems.

<ol>
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We have nothing to disclose.

多糖检测与酚-硫酸-硫酸法:不同的单糖通过展示使用这种方法9不同的最大吸收和摩尔消光系数。这导致聚合物，其含有不同量的几种糖不同的吸收最大值。吸收最大值的16不同市售聚合物不同波长在表5中给出。在聚合物中溶解（1克/升）在DDH 2 O中，搅拌（150rpm）下过夜，并用苯酚-硫酸-酸法测定。的diutan胶表现出最低的最大吸收在470nm和硬葡聚糖和透明质酸最高在484纳米。基于这些结果被选择为480nm此筛选平台。的聚合物的相对吸光度是根据用1克/升葡萄糖（设定为100％）得到的吸光度计算出来的。最低的结果，获得与核菌葡聚糖和琥珀，都与92％。这是EXPected因为硬葡聚糖只含有葡萄糖和琥珀酰聚糖含有以7:3的比例的葡萄糖和半乳糖:1。商用聚合物具有干燥和不同灰分含量的不同的损失，这就是为什么没有达到〜110％的理论值的原因。木聚糖显示出与311％的最高相对吸光度。这样做的原因是，从木糖达到的高摩尔消光系数，由于更占优势呋喃糖形式。以0.1克/升葡萄糖的水平的定量极限为止，以及在一个浓度&lt;0.05克/升的检测极限。然而，对于在筛选阳性菌株的检测极限是更高大于0.7克/升，因此，该试验显示出良好的性能。为了得到可靠的结果，凝胶过滤后剩余的葡萄糖与葡萄糖的测定法来确定，该值是从由苯酚 - 硫酸 - 酸法的值中减去。 自动葡萄糖测定稀释:性能栽培（稀释1:100）之后的葡萄糖测定进行了研究。对于这一点，将10μl的上清液在深孔板经由十倍抽吸和分配180微升出该稀释的转移到990微升DDH 2 O的和混合。第二个关键步骤是只有5从凝胶过滤后的葡萄糖测定中1:10稀释微升等分正确的移液。为了产生稀释25μl的DDH 2 O的用50微升尖首先被转移，之后20微升的DDH 2 O和5μl的凝胶滤液被一起吸出。这确保了尖端的更好除去5μl的等分试样。这两个稀释步骤通过葡萄糖测定各种葡萄糖标准进行了核实。对于两个示例性浓度，结果在表6中给出的1:100稀释的葡萄糖含量的测定后培养表明高精度用于与CV＆＃两种标准60; 1.2％。同时，对于更高的标准精度高达11.6（％误差）。然而，这是可忽略不计的葡萄糖测定仅代表培养后剩余的葡萄糖含量，因此，是不为聚合物检测重要。在1:10稀释为凝胶过滤后剩余的葡萄糖表现出非常可靠的结果与CV &lt;1.4％和精确度&lt;2.1％的误差。 考虑蒸发的:筛选需要从第一步骤到第一葡萄糖的测定3.5小时。为了找到答案，这个时间段是否有不封顶MTP存储的影响，50微升的葡萄糖检测校准标准进行储存，没有遮挡机器人传送带3.5小时。在校准范围（45到4.5毫克/升）的样品浓度几乎不增加。增加 - 引起的蒸发 - 低于2.1％和仅两个最低浓度（1.8和0.9毫克/升）则达到高达12.4％（ <str翁&gt;表7）。 凝胶过滤:高含量的非代谢葡萄糖的血糖扰乱从水解聚合物的定量检测。因此，需要凝胶过滤步骤培养后，除去剩余的葡萄糖。此外，凝胶过滤净化含有盐和单体碳水化合物的化合物，更为比葡萄糖上清液中的聚合物，以减少在该单体的分析的分析的背景。在凝胶过滤步骤35微升滤液置于井的中心。用于凝胶过滤的自动化系统的稳健性的验证，从0.045 8校准标准高达9克/升葡萄糖被过滤（N = 8）。每一浓度的葡萄糖总是由初始值（ 表8）的95％以上的降低。在这样做时，凝胶过滤显示关于各种浓度的葡萄糖的非常好的结果。另外，剩余的葡萄糖后凝胶-Filtration也与一个葡萄糖的测定法测定，并从苯酚 - 硫酸 - 酸判定减去接收葡萄糖当量的正确量的水解聚合物。 丙酮酸的测定:首先，研究了在中和和稀释（1:10）从水解步骤TFA-矩阵是否与酶反应的干扰。因此，进行了完整的测定两次，一次用和一次性无基质和显示可靠的结果。最后，16市售聚合物的丙酮酸含量成功测量并在图3中示出，它通常已知掉那些16的聚合物的仅琥珀酰聚糖和黄原天然含有丙酮酸盐。随着我们的丙酮酸法这两种聚合物被正确识别。在硬葡聚糖，在显著量也检测Welan胶和木聚糖丙酮酸盐。总体而言，该方法的能力进行了验证和丙酮酸的测定小号howed高性能。它被证明是能够水解后，以检测在不同的聚合物丙酮酸盐。 碳水化合物指纹:执行中的自动筛查所有分析模块后，被选定为碳水化合物指纹图谱分析潜在的EPS生产商。对于这一点，被应用的几个标准:1）粘度的正观测离心后和/或之后过滤。 2）沉淀过滤前后。观察纤维薄片被评价为阳性。 3）从苯酚 - 硫酸 - 酸法葡萄糖等效值。值&gt; 700毫克/升被评为积极，300和700毫克/升之间的值被评为公认的EPS生产商。当两个或三个标准被作为阳性评价，被选择用于进一步的碳水化合物指纹分析的菌株。该标准可以对EPS筛查（ 如低粘度EPS）的个人目的进行定制。我们的方法旨在寻找effici耳鼻喉科EPS生产商。当菌株只产生少量的EPS的搜索葡萄糖当量的评价极限应减少。 技术效益和未来的应用:该协议的一个有趣的特点是的步骤和不同的分析模块，模块化的特点。它们可以以不同的方式进行组合，调整至个体需要和新颖的模块可以容易地实现。此外，分析模块可以单独使用， 例如 ，在组合的水解模块对HT-PMP-衍生模块能够从在96-孔格式不同的聚合物溶液（1克/升）进行单体组合物的分析。为无需访问液体处理系统的完整筛选可以手动处理而不会在移液方案的任何改变的实验室。然而，使用液体处理系统增加了吞吐量高达768株（而不是192株如果screene手动D）每天。此处描述的协议能够为不同属的筛选，因此，对于大应变集合，以确定新的车生产者和分析其碳水化合物指纹在一个方法（ 图4）的筛选。此外，对于含有稀土糖，如岩藻糖，糖醛酸，甚至未知糖多糖靶向筛选可通过详细单糖分析进行。另外，在定义的比率不同的糖组合可以被检测出来。这使得已知的EPS或EPS新颖的结构相关的变种简单的识别。

微生物胞外多糖（EPS）是满足各种生物功能聚合物的结构高度多样化的群体。它们通常都建复杂的重复单元，其由不同类型的单体（糖，糖的衍生物，糖酸），这些单体和它们的取代之间的键区分开的。微生物多糖的结构多样性赋予这种分子类，允许类的食物1不同的领域及其应用，化妆品2,3，建筑化学4或水处理5的成员，而不同的特点。以进一步扩展这些生物基和这种可持续聚合物新颖天然微生物多糖的识别以及结构变体的工程表示有前途的方法的应用领域。无论哪种方式，需要快速筛选方法来快速扫描兵卫微生物菌种的广大- [R多糖的形成，并分析他们的产品。因此，我们最近开发了用于从天然分离或工程菌株的变体，其包括单体组合物6的识别微生物多糖生产分析的96孔HT-筛选平台。 应用此平台的第一轮筛选〜500天然分离的使我们能够确定只有约10％至20％的分离株为能够产生车（未示出数据）。这意味着，所分析的菌株的80〜90％没有产生所施加的条件下的EPS，因此，详细的碳水化合物指纹的一个进一步的分析是没有必要的。作为这种高度复杂的识别单体组合物是一个耗时的过程，特别是对于数据分析，快速的预筛选方法，以确定该菌株的EPS生产正，会急剧增加的效率。此外，试剂耗材和测量时间的UHPLC-UV-ESI-MS将会减少。此外，不同的分析模块，虽然，一方面使该方法高度可靠的，是在另一方面复杂多于两个96孔板的并联的手工处理，因此限制了该方法的全部潜力。由于这些原因，我们决定开发一个自动化筛选平台。因此，我们结合总糖含量的完全自动化的快速检测方法的现有碳水化合物指纹技术的模块化格式，根据吸光度测定。 酚-硫酸-酸方法仍然代表所选择的用于快速确定细菌和植物多糖7,8的总碳水化合物含量的方法。这种方法首先由Dubois 等人 9在96-孔板10,11描述的和适于不同的应用和样本大小，即使是小规模的。苯丙氨酸NOL - 硫酸酸方法测量一个值的总碳水化合物的含量，所有求和单体，低聚物和样品的聚合碳水化合物。 考虑到这些方面考虑，合适的培养介质的选择是必不可少的应用此方法。含低聚或类似酵母提取物高分子糖类化合物复杂的介质可能导致改变聚合物的含量，因此，应严格避免。此外，大量的糖被用作C-源菌株的培养。从栽培过程中其余的碳水化合物高水平可能负面与EPS内容的干扰测量。 因此，使用定义和纯糖的建议。在我们的实验中，我们使用葡萄糖的细胞的培养。培养后剩余的葡萄糖溶液通过凝胶过滤减少和通过葡萄糖的测定法来确定。最后，葡萄糖当量多糖第被已经用苯酚 - 硫酸 - 酸法检测到的从总碳水化合物含量凝胶过滤后减去剩余的葡萄糖计算。凝胶过滤和葡萄糖的测定与苯酚 - 硫酸 - 酸法组合保证可靠的结果，并且能够被提供了第一，完全自动化的检测系统。 两个新的分析模块，包括在自动化筛选平台，以增加从所述EPS检测系统的信息量:沉淀和粘度增大的观察。 许多不同的EPS - 例如琥珀酰聚糖，黄原胶和结肠酸12 -据报道，与糖位置C4和C6的非碳水化合物丙酮酸缩酮进行修改。那些丙酮酸缩酮（正如琥珀半酯和糖醛酸）向聚阴离子性质，因此，在物理prope通过经由二价阳离子桥13相互作用的聚合物的rties。为了确定这些特定聚合物丙酮酸的确定建立了作为另一其他分析模块。这增加了对多糖的取代基和其潜在的宏观性质的信息。 结合所有的模块使不同EPS的标识以及一个快速，高效的决心EPS生产商。通过这种方法的筛选平台可分为两个主要部分（ 图1）。在自动筛选（第一部分）的工作流中出现全自动化（ 表1）快速预选产生菌的EPS。碳水化合物指纹分析（第二部分）定量确定由预选菌株产生的EPS的单体组合物。从而，数据分析以优化的大应变集合的筛选最小化。这个提供分析在一个单一的自动筛选运行，并以二奔跑的每每天768株天是可能的384株的可能性。此外，该碳水化合物指纹分析给出了所有所识别的车的一个更详细的概述。这使定向分析和仅稍微不同的EPS的变体或完全新的识别相比EPS的已经描述的化学结构。

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="126">
			<td height="126" style="height:126px;width:335px;">96 well deep well plate 2.0 mL (DWP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">780271</td>
			<td style="width:224px;">Main-culture (CP 1-1 to 1-4), equilibration plates for gel-filtration (CP 1-6, 1-7, 2-6 and 2-7), 1:100 dilution for the&#160; glucose assay (CP 4-1 to 4-4) containing 990&#181;l of ddH2O</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Breathable Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">BF-400-S</td>
			<td style="width:224px;">Incubation film for pre- and main-culture DWP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Aluminum Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">PCR-AS-200</td>
			<td style="width:224px;">-80 &#176;C storage film for glycerol-stock plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">MCA96 Nested Disposable Tips 200 &#181;l</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038619</td>
			<td style="width:224px;">Worktable position (WTP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">A/B glass-filter-plate 1&#181;m</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8031</td>
			<td style="width:224px;">Stored on collector plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">651201</td>
			<td style="width:224px;">Collector plate for filtration plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well SpinColumn G-25</td>
			<td style="width:185px;">Harvard Apparatus</td>
			<td style="width:143px;">74-5612</td>
			<td style="width:224px;">Stored on washing DWP (CP 1-6, 1-7, 2-6 and 2-7)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:143px;">249944</td>
			<td style="width:224px;">Collector plate for gel-filtration plate (CP 3-1 to 3-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Nested Disposable Tips SBS 50 &#181;l tips</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038609</td>
			<td style="width:224px;">Carousel position (CP 4-6, 4-7 and 5-1 to 5-6)</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:335px;">Trough 250 ml</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">Res-SW96-HP</td>
			<td style="width:224px;">Water WTP 1-1, Glucose assay reagent-mix WPT 1-2, ammonium-acetat buffer pH 5.6 (CP 5-7)</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:335px;">96-well micro titer plate F-Bottom (MTP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">655101</td>
			<td style="width:224px;">precipitation 1 (CP 6-1 to 6-4), pH-value (CP 7-1 to 7-4), precipitation 2 (CP 8-1 to 8-4), phenol-sulfuric-acid method (CP 8-5 to 8-8), glucose-assay (CP 9-1 to 9-9), dummy plates (WTP 3-1, 3-2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">96-well silicon cap mat&#160;</td>
			<td style="width:185px;">Whatmann</td>
			<td style="width:143px;">7704-0105</td>
			<td style="width:224px;">Cover mat for MTP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">200 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.760.002</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1000 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.762</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well-PCR micro titer plate&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781350</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">TPE (thermoplastic elastomer) cap mat&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781405</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:335px;">Filter plate 0.2&#160;&#181;m Supor,&#160;</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8019</td>
			<td style="width:224px;">Filtration of samples for UHPLC-ESI-MS analysis with a MTP collector plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pipette Tips LHS 5-300 &#181;l</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">732150</td>
			<td style="width:224px;">Brand LHS system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">ABTS (2.2-azino&#8209;bis-(3&#8209;ethylbenzthiazoline)-6-sulfonic&#160;acid)</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">A1888</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Glucose oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">G2133</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Horseradish peroxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6782</td>
			<td style="width:224px;">Glucose-assay, pyruvat-assay</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:335px;">DA-64 (N-(Carboxymethylaminocarbonyl)-4.4&#39;-bis(dimethylamino)-diphenylamine sodium salt)</td>
			<td style="width:185px;">Wako Chemicals GmbH</td>
			<td style="width:143px;">043-22351</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pyruvate oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4591</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate dibasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P749.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate monobasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">3904.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Thiamine pyrophosphate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">C8754</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Magnesium chloride hexahydrate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">31413</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">2-Propanol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20922.466</td>
			<td style="width:224px;">Precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Phenol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20599.231</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Sulfuric acid</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">4623.4</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Trifluoroacetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">T6508</td>
			<td style="width:224px;">Hydrolysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ammonium solution</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P093.1</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Phenol red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">B21710</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1-Phenyl-3-methyl-5-pyrazolone</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">M70800</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methanol LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83638.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetonitril LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83040.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">338826</td>
			<td style="width:224px;">PMP-derivatization,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methyl red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">36667</td>
			<td style="width:224px;">pH-value</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Robotic liquid handling system&#160;</td>
			<td>Tecan</td>
			<td>Freedom EVO</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid handling station LHS</td>
			<td>Brand</td>
			<td>709400</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip-Adapter&#160;</td>
			<td>Brand</td>
			<td>709434</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Ends MC 20-300&#181;L</td>
			<td>Brand</td>
			<td>709416</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adapter 60mm</td>
			<td>Brand</td>
			<td>709430</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
	</tbody>
</table>

automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis

许多微生物能够产生和分泌的胞外多糖（EPS），其具有在医学领域，食品应用或在更换的基于石油化工的重要影响。我们描述了一种液体处理系统，其允许的类型和EPS的由微生物产生的量的快速和可靠的分析，以进行自动化分析平台。它使用户能够确定新的天然微生物胞外多糖生产者和分析在一天之内的相应聚合物的碳水化合物指纹在高吞吐量（HT）。使用此平台，应变集合以及可能在工程方法来获得的菌株的变体的文库可以筛选。该平台有一个模块化设置，其允许分离协议的为两个主要部分。首先，有一种自动筛选系统，它结合了不同的多糖的检测模块:viscosit的半定量分析通过离心步骤，通过乙醇沉淀的聚合物的形成进行分析，并通过苯酚 - 硫酸 - 酸转化的总碳水化合物含量的测定ý形成。这里，有可能筛选高达每运行384菌株。第二部分通过组合两个基本模块为所有在第一部分中确定的选定车生产者详细单糖分析:完整单体组合物通过超高效液相色谱加上紫外线和电喷雾电离离子阱检测（分析UHPLC-UV-ESI-MS）和丙酮酸的确定为耦合到一个颜色形成通过酶促氧化的聚合物取代基（丙酮酸缩酮的存在下）。所有这种筛选平台的分析模块可以以不同的方式进行组合，并调节至个别的要求。此外，它们都可以被人工处理或与液体处理系统进行的。由此，筛选PLATForm的实现了巨大的灵活性，以确定不同的EPS。

Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

automated-modular-high-throughput-exopolysaccharide-screening

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

11.4K 次观看.  Technische Universität München. We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

视频: Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...