作者感谢托马斯博士拉特马赫提供的PPAM植物表达载体和易卜拉欣Amedi培养在这项研究中所使用的烟草植株。我们要感谢理查德·泰曼博士的协助编辑稿件。这项工作是部分资金由欧洲研究理事会高级格兰特“未来医药”，提案编号269110和Fraunhofer Zukunftsstiftung（弗劳恩霍夫未来基金会）。

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该刊物的费用部分由公司Statease公司（美国）及STATCON（德国），这些都没有涉及参与的手稿或承担任何其内容的准备赞助。

每个实验需要仔细的规划，因为资源通常是稀缺和昂贵。这是能源部战略尤其如此，因为在规划阶段（ 如选择基本模型，它并不包括所有显著因素相互作用）的错误可以大大减少所产生的模型的预测能力，从而贬低了整个实验。然而，可以很容易地通过以下方式避免基本程序这些错误。 在能源部的规划注意事项首先，要选择一个响应，适合于评估结...

生物的蛋白质在植物中的产量是有益的，因为植物是便宜的增长，该平台可以扩大刚刚通过生长得更植物和人类病原体无法复制1,2。例如基于与叶根癌农杆菌浸润瞬时表达策略提供了额外的好处，因为DNA传递和净化产品的交付点之间的时间减少从几年到不到2个月3。瞬时表达也可用于功能分析， 例如来测试基因的互补功能丧失的突变体或以调查蛋白质相互作用4-6能力。然而，瞬时表达水平往往表现出更大的批与批之间的变化比在转基因植物中7-9的表达水平。这减少了基于瞬时表达的Wi生物制药生产过程的可能性会批准在良好生产规范（GMP）的情况下，因为再现性是一个关键质量属性，并受风险评估10。这种变化也可以掩盖，研究人员打算调查的任何相互作用。因此，我们开始着手找出影响植物中瞬时表达水平，打造一支高素质的定量预测模型的主要因素。 一个因子在每次一个（OFAT）方法经常用于表征某些参数（ 系数 ）的对结果的实验11的影响（ 效应 ）（ 响应 ）。但是，这是不理想的，因为单独的测试（ 运行 ）的调查（ 实验 ）期间将通过被测试（ 设计空间 ）的因素跨越的潜力区对齐像一串珍珠。设计空间和信息，因此从实验中得出的程度的覆盖范围是低， 如图1A 12。此外，不同的因素（ 因子相互作用 ）之间的相互依赖关系可以保持隐蔽导致差的模型和/或假最优的预测， 如图1B 13。 上述缺点，可避免使用试验设计（DOE）的方法，其中一个实验的运行被整个设计空间散布更加均匀地设计，这意味着多于一个因素是两个运行14之间变化。有专门设计的混合物，筛选因子（ 因子设计 ）和对反应的影响因素的定量（ 响应面方法，RSM次）15。此外，的RSM可被实现为中央复合设计，但也可以通过使用能够对运行中的选择应用不同的标准专门的软件有效地实现。例如，所谓的D-optimalitŸ标准将选择运行，以便在生成的模型的系数，以尽量减少误差，而IV-最优标准选择运行的实现整个设计空间15,16最低的预测方差。我们在这里描述的RSM允许在植物中瞬时表达的精确定量，但是它可以很容易地转移到涉及多个（〜5-8）的任何系统的数值的因素（ 如温度，时间，浓度）和几个（〜2 - 4） 明确的因素（ 如启动子，颜色），其中一种机械的描述不可用或过于复杂的模型。 能源部的方法起源于农业科学，但已经扩散到其它区域，因为它是可转移到任何情况下它减少必要获得可靠的数据运行的次数，并生成描述性模型为复杂的过程是有用的。这反过来又导致了“指引纳入能源部的业，Q8（R2）医药发展“通过对技术要求国际协调会议公布了注册人用药品（ICH）17。能源部目前广泛应用于科研和工业18。然而，护理过程中必须采取规划和执行实验，因为选择不当的多项式程度的多重线性回归模型（ 基本型号 ）可以引入一个需要额外的运行，以正确模拟所有因素的影响。此外，损坏或丢失的数据产生不正确的型号和有缺陷预测，甚至可能防止在协议和讨论第18条所述的任何模型建立的尝试。在协议中，我们将首先列出最重要的规划步骤为RSM为基础的实验，然后解释根据能源部的设计软件DesignExpert V8.1，但类似的设计可以与其他软件includi建吴JMP，Modde和STATISTICA。实验步骤之后是对数据进行分析和评价的指示。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig1highres.jpg" src="/files/ftp_upload/51216/51216fig1.jpg" /> 图1。 OFAT和能源部，一个比较 。的一个因素在一个实验（黑，红和蓝色圆圈）连续变化的时间（OFAT）实现了设计空间（斜线区域）的低覆盖率。与此相反，一个以上的因素在一个时间使用的试验设计（DOE）的策略（绿色圆圈）的设计的变化提高了覆盖率，将所得模型。 乙因而精度。偏置设计空间覆盖意味着OFAT实验（黑圈）也可能失败，找出最佳的操作区域（红色）和预测次优的解决方案（大黑圆），而能源部将军会ES（黑星）更容易找出最好的条件（大黑星）。

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td style="width:272px;">DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tryptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8952.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2363.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ampicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">K029.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agar-Agar</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">5210.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Escherichia coli K12 DH5a</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">18263-012</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">pPAM</td>
			<td style="width:195px;">GenBank</td>
			<td style="width:119px;">AY027531</td>
			<td>Cloning/expression vector;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Plasmid&#160;</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740588.250</td>
			<td>Plasmid DNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Gel and PCR Clean-up</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740609.250</td>
			<td>Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NanoDrop 2000</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">n.a.</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NcoI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3193L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EcoRI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3101L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">AscI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R0558L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NEB 4</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">B7004S</td>
			<td>Restrictionendonuclease buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TRIS</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4855.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium tetraborate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4403.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8040.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6352.4</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bromophenol blue</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A512.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Xylene cyanol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A513.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glycerol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">7530.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Mini-Sub Cell GT Cell</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">170-4406</td>
			<td>Gel electrophoresis chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agrobacterium tumefaciens strain GV3101:pMP90RK</td>
			<td style="width:195px;">DSMZ</td>
			<td style="width:119px;">12365</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Electroporator 2510</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">4307000.658</td>
			<td>Electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Beef extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">X975.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Peptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2365.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sucrose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4621.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magnesium sulfate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">0261.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Carbenicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6344.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Kanamycin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">T832.3</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Rifampicin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4163.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FWD primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCT CAG GAA GAG CAA TAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">REV primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCA AAG CGA GTA CAC AAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2720 Thermal cycler</td>
			<td style="width:195px;">Applied Biosystems</td>
			<td style="width:119px;">4359659</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">RNAfold webserver</td>
			<td style="width:195px;">University of Vienna</td>
			<td style="width:119px;">n.a.</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">n.a.</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Omnifix-F Solo</td>
			<td style="width:195px;">B. Braun</td>
			<td style="width:119px;">6064204</td>
			<td>Syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Murashige and Skoog salts</td>
			<td style="width:195px;">Duchefa</td>
			<td style="width:119px;">M 0222.0010</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glucose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6780.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acetosyringone</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">D134406-5G</td>
			<td>Phytohormon analogon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">&#160;BioPhotometer plus</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">&#160;6132 000.008</td>
			<td>Photometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">n.a.</td>
			<td>SPR device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Protein A</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">10-1006</td>
			<td>Antibody binding protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tween-20</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9127.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2G12 antibody</td>
			<td style="width:195px;">Polymun</td>
			<td style="width:119px;">AB002</td>
			<td>Reference antibody</td>
		</tr>
	</tbody>
</table>

characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study

工厂提供用于生产生物制药，包括成本低，可扩展性和安全性的多重效益。瞬时表达提供短期开发和生产时间的额外优势，但表达水平可以分批从而在良好生产规范的背景下监管问题引起的显著差异。我们使用的试验设计（DOE）的方式设计确定的主要因素，如表达过程中的表达构建体的调节元件，植物生长和发育的参数，并在培养条件下，在表达的批次之间的变异性的影响。我们测试了植物表达模型的抗HIV单克隆抗体（2G12）和荧光标记蛋白（红色荧光蛋白）。我们讨论的理由来选择模型的某些性质，并确定其潜在的局限性。一般的处理方法可以很容易地转移到其他问题，因为该模型的原理再广泛适用：基于知识的参数选择，降低复杂性通过把最初的问题分成更小的模块，优化实验组合软件引导安装程序和阶梯式设计增强。因此，该方法不仅用于表征植物蛋白的表达也为其他复杂系统缺乏一种机械描述的调查有用的。预测方程描述参数之间的互连性可用于建立机理模型用于其它复杂的系统。

使用不同的启动子和5&#39;UTRs过程中瞬时表达红色荧光蛋白积聚的描述性模型 在叶中提取红色荧光蛋白的荧光被用来表明该重组蛋白的表达水平，并因此被用作在能源部策略的响应。我们认为相关的最小可检测差异为20微克/毫升和系统的估计的标准偏差为8微克/毫升的基础上初步实验。包含在瞬时表达模型的因素的基础上文献资料7,8和我们以前的结果9...

Watch this Scientific Journal Video about Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study at JoVE.com

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

我们描述的实验方法设计，可以被用来确定与模型的转基因的调控元件，植物生长和发育参数和培养条件对植物中的单克隆抗体和报告蛋白的瞬时表达的影响。

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

characterization-complex-systems-using-design-experiments-approach

Research

JoVE Journal

Bioengineering

16.4K 次观看.  RWTH Aachen University. 我们描述的实验方法设计，可以被用来确定与模型的转基因的调控元件，植物生长和发育参数和培养条件对植物中的单克隆抗体和报告蛋白的瞬时表达的影响。 

视频: Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

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 ...

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. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

1型糖尿病生物制药候选疫苗在红甜菜中的瞬态表达

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

三尖瓣瓣叶的层显微切割，用于双轴力学表征和微观结构定量

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...