这项工作是由美国能源部能源大湖生物能源研究中心"（能源部办公室科学的BER DE - FC02 - 07ER64494），并授予DE - FG02 - 91ER200021中的一部分，美国能源部基础能源科学，科办公室化学科学，地球科学和生物科学。我们感谢他们的物质和概念贡献的John Scott Craig和梅丽莎Borrusch。

1。产酶<ol><li> 在毕赤酵母中的蛋白质生产到向量pPICZ或pPICZα相应基因的克隆和改造成体育酵母 。在百思不得其解的500毫升烧瓶增长甲醇诱导细胞在300毫升的批次，每24小时（班纳吉等人，2010A）。 </li><li>精矿和切向流过滤脱盐培养滤液。 </li><li>商店小等分的酶在20％甘油于-80 ° C。毫克/毫升的浓度范围是1-10。 </li></ol> 2。试验设计<ol><li>输入要测试的酶的数量，他们的上限和比例较低（例如一个核心酶的5％的比例较低）和实验设计的类型（例如，二次或三次）到设计，Expertsoftware。 </li><li>计算微克每一个要加入使总负载的15毫克/克葡聚糖酶的数量，然后计算出每一种酶在μL的量添加到每个反应。 </li><li>生成Excel工作表指定配药水和酶的实验设计和使用从文件转移功能导入Biomek的FXP软件源的实验器皿，井源，目的地实验室器皿，目的地水井，和传输卷。 </li></ol> 3。酶解<ol><li>暂停50 mM柠檬酸钠，pH值4.8生物质的泥浆，含5μg/ ml的四环素和5微克/毫升酮，在桨水库。 </li><li>机器人吸管的泥浆200μL到每孔96孔深孔反应板采用跨度8 1000 -μL与去年0.5厘米切断的吸管提示。一次混合的泥浆，在100μL/秒，然后吸桨水​​库相同体积和分配到板。 </li><li>免除到同一板使用的程序输入到贝克曼FX的酶。 </li><li>穿孔capmats密封板。 </li><li>倒像，挖掘板块多次克服表面张力。杂交孵化器旋转10转48小时，放置在50 ° C的板块。 </li></ol> 4。 GLC和木糖的测定<ol><li>摆动桶离心机在1250 XG 3分钟离心反应板。 </li><li> 100μL上清转移到常规96孔板使用的Biomek FX AP96 POD。 </li><li>孵育板在90-95 ° C下10分钟，以灭活酶，然后在1250 XG离心30秒。 </li><li> GLC测量，转移到常规的96孔板12μL上清。加入192μL的葡萄糖氧化酶/过氧化物酶（GOPOD）试剂。板在50 ° C孵育20分钟，并宣读酶标仪在510 nm处的吸光度。空白是没有酶反应混合物。 </li><li>木糖测量，转移到384孔板4μL。加入检测试剂，并在340 nm处读取的板块。空没有酶的反应混合物。 </li></ol> 5。数据分析<ol><li>转换的吸光度值％GLC和木糖产量的基础上著名的GLC和木糖的原生物质的含量。这些百分比作为回应导入到设计专家软件。检查统计paramenters，以确保一个强大的模型的标准履行。 </li><li>确认实验的最佳模型预测。 </li></ol> 6。与GENPLAT代表性的成果: （1）建立个人纯蛋白质的优化混合物 。结果表明酶是重要的，以及在什么比例，而其中的酶并不重要。一些蛋白质丰富的T.瑞氏木霉 secretome（Nagendran等，2009），如Cip1和Cip2，玉米秸秆预处理氨纤维膨胀（AFEX）或碱性路政署没有作用发挥GLC释放<html> rogen过氧化氢法（AHP）（班纳吉等人，2010B）。另一方面，一些蛋白质是出乎意料的重要。例如，内切木聚糖酶糖基水解酶家族10日和11日都是重要的GLC释放一个木聚糖酶不能为其他（班纳吉等人，2010B，C）代替。 Cel61A，轻微的蛋白质中的T.瑞氏木霉 secretome，是非常重要的GLC，但不木糖释放。某些酶的重要GLC和木糖的释放，而另一些仅适用于一个或另一个（班纳吉等人，2010C）的必要。 16个组件，每一个合成的混合物，含有浓度为0.94毫克/毫升（即非优化的混合物），发布从AFEX预处理的DDG - 38％可用GLC。在相同的条件下水解下，优化的混合物，其中的16个组件的浓度范围从0％到32％，52％的GLC公布（图2）。这个实验说明了建设定义混合物的效用。 _content"&gt;（2）建立不同的预处理/基材组合优化的鸡尾酒。当前商业纤维素酶制剂是"一个尺寸适合所有"，在现实中，最好的鸡尾酒取决于基板和预处理。单一产品如Accellerase 1000提供了从不同的基板，以同样的方式预处理，从接触到不同的预处理（图1）在同一基板不同的收益率。我们用GENPLAT优化多个预处理和多发原料（班纳吉等纯酶的混合物人，2010C）。图2显示了如何AFEX预处理玉米秸秆，AHP预处理玉米秸秆，AFEX预处理DDG - 16纯酶最佳酶的比例不同。只有11酶在图2所示，因为最佳被发现的其他5个（Cel61B AbfB，Cip1，Cip2，并Cel12A）的比例为0​​％（班纳吉等人，2010C）。 （3）新型酶的发现 </strONG&gt;。的纯蛋白质的优化混合物的建设是一个持续的过程。由于新的蛋白质成为纯粹的形式（从商业来源，异源表达，或净化）提供的，他们可以将它们添加到当前的优化设置，系统测试。 GLC或木糖的新型蛋白质，有助于释放，然后成为一个新的，优化组合的一部分。这样，GENPLAT变成一个确定新的酶生物质解构平台。我们已经取得了其他真菌提取物比t 瑞氏木霉和A 。 尼日尔不同衬底上生长。加入到我们的核心设置一个特别提取带动GLC产量。负责蛋白质纯化，并没有任何商业酶制剂（Banerjee和沃尔顿，未公布结果）是一种新的糖基水解酶。 （4）发现更好的酶 。显示GENPLAT哪些酶是最重要的的一个退化特别是生物质，我们有更好的理解其中的酶应重点改善。可以找到更好的关键酶的例子在自然界寻找或蛋白质工程。 （一种酶可以"更好"比T.瑞氏木霉同源取决于所需的属性，在几个方面，例如，更高的具体活动，更大的协同作用，减少蛋白水解的灵敏度，或热稳定性增强）。 GENPLAT，提供了一个有意义的化验可能更好的酶平台，因为它采用了切实复杂的鸡尾酒（重要，因为没有生物酶隔离工程）和一个现实的基板（重要的，因为纯纤维素或其他人造基板的结果可能不相关天然木质纤维材料）。 （5）商业酶的优化 。纯酶的数量足够大的还没有在现实世界中存在。直到他们这样做，乙醇亲ducers必须依靠商业产品，如Accellerase 1000和MultifectXylanase。然而，商业酶的混合物，通常执行比任何个人的更好，GENPLAT可用于设计多个商业酶的优化鸡尾酒。图3显示了GENPLAT使用优化的四种商业酶制剂的混合物。 AFEX预处理DDG - GLC释放的最佳比例分别为47％Accellerase1000 Multifect果胶酶Novozyme 188，22％和4％Multifect木聚糖酶（班纳吉等人，2010C），27％。图3b显示AFEX - DDG - GLC产量从四个酶制剂的差异;优化的商用混合物提供了更高的GLC产量仅比Accellerase1000，SpezymeCP单独，或16组件的合成混合物。这个实验还表明，16组件的合成混合物是缺少一个或多个酶最佳GLC释放，这是在一个或更多的商业酶需要。 GENPLAT可以被我们教育署，以确定这样的组件。 <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="图1"/> 图1。没有一个单一的商业酶制剂是所有基材和所有预处理的最佳选择。五基板和三个预处理磨（0.5毫米粒度）的类似条件下进行比较，酶量（15毫克/克葡聚糖）和水解条件（48小时，50 ° C）A.不同的AFEX预处理基板消化Accellerase 1000。B.消化玉米秸秆预处理Accellerase 1000由三个不同的方法（见班纳吉等人，2010C）。 <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="图2"/> 图 11 AFEX预处理玉米秸秆（AFEX - CS），碱性过氧化氢 ​​预处理玉米秸秆法（AHP - CS），或AFEX预处理干GLC释放酶的最佳比例提取2 。ERS"五谷（AFEX副总干事）。班纳吉等人的数据。 （2010C）。沿的酒吧上方的数字是在生物样品的总GLC％GLC产量。 <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="图3"/> 图3。GENPLAT使用产生四个AFEX预处理DDG - GLC发布的商业酶制剂的优化混合物。 Multifect木聚糖酶的贡献最小（4％），因此，从这个三元相图省略。B.从AFEX DDG - GLC产量与在相同的条件下水解（15毫克/克葡聚糖酶，48小时，50℃）不同的酶处理。 "四组分商业"已确定的比例从如图所示的实验。 3A。

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没有利益冲突的声明。

人们普遍认识到，降低酶的成本经济的木质纤维素乙醇产业的发展是很重要的。目前可用的商业酶鸡尾酒复杂不清的许多蛋白质​​（Nagendran等人，2009年）的混合物，他们主要是为适应酸预处理的玉米秸秆使用。为了加快发展更好的酶鸡尾酒，一些实验室已经开发出高通量平台，酶的发现和鉴定。在这方面的努力已纳入也发现在GENPLAT以下属性的一个或多个:配药机器人的酶和生物量的泥浆，统计，实验设计，和/或GLC自动化的决心和木糖（柏林等，2007;德克尔等。人，2009年;金等，1998; King等，2009）。 GENPLAT扩展这些早期的努力，最显著最多6个组件，可以从分析的复杂性，在酶的混合物早期的研究在我们的最新作品，以超过16（班纳吉等人，2010C）。 GENPLAT其它关键特性是珠混合室（桨水库）的使用，可以保持在配药暂停秸秆泥浆;温柔消化在年底高端旋转混合;和自动化比色法测定谷氨酸和木糖。

用于设备和软件: <ol><li>设计专家软件，8.0版（STAT - EASE，公司，明尼阿波利斯，明尼苏达）（ <a href="http://www.statease.com">http://www.statease.com</a> ） </li><li> Biomek FXP的实验室自动化工作站（贝克曼 - 库尔特，富勒顿，CA） </li><li> Biomek的FXP软件，版本3.3（贝克曼 - 库尔特，富勒顿，CA） </li><li>桨水库（型号756C - 1P100副总裁，五普科技公司，圣地亚哥） </li><li>板，96深（1.5毫升）（Fisher Scientific这Abgene，爱普生，英国） </li><li> （佛罗里达州奥卡拉，美国科学，穿孔capmats） </li><li>旋转杂交培养箱（型号5420，VWR） </li><li>摇摆桶离心机（型号5810R，Eppendorf公司，汉堡，德国） </li><li>板，中光学96（Abgene尔科技，爱普生，英国） </li><li>板，384孔（BD，BD公司，加利福尼亚州圣何塞，猎鹰） </li></ol>所用试剂: <ol><li> 毕赤酵母载体:应变X33，pPICZ和pPICZα（Invitrogen公司，卡尔斯巴德，CA） </li><li> T.瑞氏木霉和向量:应变QM9414（美国农业部国家农业应用研究中心，皮奥里亚，白细胞介素部），pCB1004 - EV（班纳吉等人，2010B。） </li><li> Accellerase 1000（批号:1600844643）（杰能科公司，罗切斯特，纽约） </li><li> 50 mM柠檬酸钠缓冲液，pH值4.8 </li><li>四环素类股票:10毫克/毫升</li><li>放线菌酮的股票:10毫克/毫升</li><li> GOPOD测定试剂盒，目录的K - GLUC（Megazyme，布雷，爱尔兰） </li><li>木糖测定试剂盒，目录的K -木糖（Megazyme，布雷，爱尔兰） </li></ol>

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

酶的生物质解构的成本高，木质纤维原料的液体运输燃料如乙醇的经济转换的一个主要障碍。我们已经开发了一个集成的高通量平台，为来自不同的预处理/生物组合的糖释放的发现和开发新的酶和酶鸡尾酒GENPLAT。 GENPLAT包括四个要素:个人纯酶，统计设计的实验，生物泥浆和酶的机器人pipeting，并自动发布GLC和木糖colorimeteric决心。个人酶在毕赤酵母或里氏木霉 ，中的表达，或从商业鸡尾酒或新型微生物提取物的色谱净化生产。单纯格（部分因子）混合模型设计商业设计实验统计软件的使用。高度复杂的酶混合物性构造成96格式的机器人pipeting。发布GLC和木糖的测量是采用酶联比色法自动化。已经过测试，对各种原料和预处理组合优化酶的混合物，含有多达16个组件。 GENPLAT是适应纯酶，商业产品的混合物（例如，1000 Accellerase和Novozyme 188），新型微生物的提取物，或组合的混合物。为了使测试〜10纯酶的混合物，要求小于100微克每一个蛋白质和少于100人的总反应，蛋白质15毫克/ G葡聚糖在最后的总负荷运行时。我们使用从多个来源的酶。纯化的酶可以从天然来源，如真菌文化（如黑曲霉（Aspergillus niger），Cochliobolus carbonum，Galerina marginata），或他们可以通过编码基因的表达（从增加numbe获得R的微生物基因组序列）在诸如E.主机大肠杆菌，毕赤酵母 ，或如T.丝状真菌瑞氏木霉 。蛋白质也可以从商业酶鸡尾酒（例如，Multifect木聚糖酶，Novozyme 188）的纯化。越来越多的纯酶，包括糖基水解酶，细胞壁积极酯酶，蛋白酶，和裂合酶，可从商业来源，例如，Megazyme公司（www.megazyme.com），NZYTech（www.nzytech.com） PROZOMIX（www.prozomix.com）。 设计专家软件（STAT - EASE，公司）是用于创建单纯晶格的设计和分析响应（在这种情况下，GLC和木糖发布）。混合物含有4-20组件，它可以在0和100％之间的比例不同。检测点通常包括足够数量的干预点，以产生一个有效的模型的极端顶点。在实验设计的术语，我们的研究大多是"混合"的实验，这意味着一笔将组件添加到一个全社会固定资产蛋白加载（毫克/克葡聚糖表示）。单纯晶格的混合物的数量依赖于混合物中的元件数目和多项式（二次或三次）的程度。例如，6组件的实验将需要63增强特殊的立方模型不同的反应，它可以检测三个双向互动，而只有23个人的反应是必要的增强二次模型。对于包含超过八个组成部分的混合物，二次实验设计更实用，而且在我们的经验，这种模型通常是统计学上有效的。 所有的酶负荷，最后总负荷（我们的实验通常是15毫克蛋白质/ G葡聚糖）的百分比表示。对于"核心"的酶，较低的百分比限制设置为5％。此限制是来自我们的经验，GLC和/或木糖的产量非常低，如果任何核心酶是目前在0％。不良模型结果从太多的样品表现出非常低的GLC或木糖产量。依次设置下限，确定一个上限。也就是说，一个六分量的实验，如果设置为5％的下限为每个单独的组件，然后将各单项组件的上限75％。被视为"附件"的所有其他酶的下限设置为0％。 "核心"和"附件"的名称有点乱，将基板上的不同而有所不同，但在我们的研究从玉米秸秆释放GLC的核心酶包括以下酶从T 瑞氏木霉 :CBH1（也被称为Cel7A），CBH2（Cel6A），EG1（Cel7B），BG（β-葡萄糖苷酶），EX3（远藤-β1,4 -木聚糖酶，GH10），和BX（β-木糖苷酶）。 <div id="myEventWatcherDiv"></div>

Watch this Scientific Journal Video about GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization at JoVE.com

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT（GLBRC酶纲要"）是一个生物质降解酶鸡尾酒的发现和优化的自动化平台。它可适应多种原料和包含多个组件的酶的混合物。

GENPLAT:生物质酶的发现和鸡尾酒优化的自动化平台

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Research

JoVE Journal

Bioengineering

15.4K 次观看.  Michigan State University. GENPLAT（GLBRC酶纲要"）是一个生物质降解酶鸡尾酒的发现和优化的自动化平台。它可适应多种原料和包含多个组件的酶的混合物。

视频: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

FR-4 铜包电极板导电微泵性能测试平台

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...