Name: fibs-enabled noninvasive metabolic profiling
Uploaded: 2014-02-03T15:15:00
Description: Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

我们感谢狼Frommer教授（卡内基科学研究所，斯坦福大学）的好心为我们提供的葡萄糖FRET质粒和乌韦Ludewig博士（霍恩海姆大学）的亲切供应谷氨酰胺FRET构建在pUTKan植物表达载体。 AB公司是由BBSRC针对性的优先级奖学金计划提供资金。既CK和KP是由英国研究理事会奖学金的生物制药工艺的支持。 CK还要感谢龙沙生物提供财政支持。该中心为合成生物学与创新是慷慨由EPSRC支持。

1。细胞系的复兴和维护<ol><li>恢复在补充有8 2mM L-谷氨酰胺和10ml / L的100倍的次黄嘌呤/胸苷补充物（完全培养基）9毫升的CD-CHO培养基中的CHO细胞。 </li><li>离心机在100×g离心5分钟。 </li><li>重悬在10毫升新鲜的完全生长培养基中的细胞。 </li><li>取出1ml样品，用光学显微镜测定的活菌浓度使用台盼蓝染料排除法用血细胞计数。 </li><li>启动培养以3×10 5细胞/在125毫升毫升的接种密度摇瓶。 </li...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. 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该FIBS使在体内和关键分子原位定量，在这种情况下生长限制性营养物质，除去从淬火和提取方法所产生的不确定性。调查结果表明，有在FIBS信号和细胞内浓度在1-5毫米的范围内葡萄糖和0.3-2mm的谷氨酰胺之间的良好关系。在批量的CHO细胞培养中，这些浓度中的指数，固定的，并且早下降阶段遇到的问题。该指数相和固定相是最关键的适宜喂养策略的设计方面，因此使用这些生物传感?...

代谢物监测在生物加工各种应用，包括工艺开发，媒体和饲料的设计，以及代谢工程。各种方法可用于浓度测量通过使用酶1，化学2，或结合试验3。一个有趣的选项是使用荧光，蛋白质为基础的体内生物传感器作为关键代谢产物的细胞内浓度的记者。在超低量测定法，荧光是一种方便的工具，如小型化实际上是改善了信号-噪声比4,5和蛋白为基础的传感器可遗传编码的意义，没有外源试剂是必需的代谢物的分析。福斯特共振能量转移（FRET）生物传感器包括一个配体结合域连接的两个荧光蛋白。 FRET是能量从光激发捐助者的一个非辐射转移到受体荧光分子要价较高，<html>编接近（&lt;100）。配体的裂解或结合引起的构象变化的传感器，其中，反过来，诱导的荧光基团的接近而变化，导致通过在发光光谱的变化测定FRET效率的变化。 FRET集成生物传感器（FIBS）的细胞系中是组成型产生的和它们的光谱特性改变的基础上代谢物在细胞内的浓度。 FIBS具有快速响应时间使它们非常适合进行测量的动态模型6。以前的应用包括监测单7-9和10多个代谢物，以及提供有关时空分布11个数据。载脂蛋白最大，其中配体结合打乱降低能量传递和载脂蛋白民，其中配体结合所带来的两个荧光基团成更紧密的接触（ 图1）的荧光团的接近：FIBS可以在两种配置中创建。 _content“&gt;在这项工作中，一个协议，提出构建中国仓鼠卵巢（CHO）细胞系中是组成型表达一个FIBS为代谢物，葡萄糖或谷氨酰胺，一种方法是建立在传感器体 ​​内的校准，以使将来的定量胞内代谢物浓度的测量，如示于图2。基于此，葡萄糖或谷氨酰胺的细胞内浓度可在补料分批的CHO细胞培养物测定中，向其中两种养分中加入高浓度的过程，其结果证明由FIBS和先前构建的校准曲线使用的荧光信号，准确预测细胞内浓度是可能的，并经一个独立的酶法测定，该方法提供了超过当前的分析技术显着的优点，因为它是无创的，低成本的快，给，可在星期一FIBS的实时信号itored整个文化。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

在计算生物学的时代，新的高通量实验系统是必要的，以便填充和完善的模型，使他们能够进行预测进行验证。理想的情况是这样的系统将是低量，这就排除了采样和破坏性的分析时，时间过程的数据将被获取。所需要的是一种在原位监测工具，它可以在实时和非侵入性地报告必要的信息。一个有趣的选项是使用荧光，蛋白质为基础的体内生物传感器作为细胞内浓度的记者。已经发现应用中代谢物的定量一个特定类的体内生物传感器是基于共振能量转移（FRET）的配体结合结构域连接的两个荧光蛋白之间。 FRET集成生物传感器（FIBS）细胞系中都产生组成，他们有快速的响应时间和他们的茶谱根据代谢物在细胞内的浓度特性的研究改变。在本文中，该方法用于构建中国仓鼠卵巢（CHO）细胞系中是组成型表达一个FIBS为葡萄糖和谷氨酰胺和校准体内批次细胞培养物中FIBS以使胞内代谢物浓度的将来量化进行说明。从进料 - 分批的CHO细胞培养数据表明，FIBS能够在每种情况下，检测在细胞内的浓度所产生的变化。使用从FIBS和先前构建的校准曲线的荧光信号，细胞内浓度，精确地确定所确认的独立酶法测定。

该方法的概述示于图2。在这里提出的工作，CHO细胞用的FIBS载体和稳定细胞系，在400微克/毫升硫酸博来霉素的抗生素压力被选定。两个独立的稳定细胞系组成性表达的葡萄糖和谷氨酰胺的传感器因此被创建。 图1描述了在本研究中使用的生物传感器2的配置。葡萄糖传感器是基于载脂蛋白 - 最大原理和相应的质粒是PRSET-FLIPglu600μΔ11Aphrodite。这是一种融合增强型青色荧光蛋?...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

如何在原位代谢轮廓校正共振能量转移集成生物传感器（FIBS）用于描述呈现。该FIBS可用于测量非侵入性代谢物辅助的代谢模式和生物过程条件的高通量筛选的发展的细胞内水平。

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Research

JoVE Journal

Bioengineering

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

原位压极限和骨牙周韧带齿纤维联合的相关性无创成像

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Calorespirometry: 一种功能强大、无创的研究细胞能量代谢的方法

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...