Name: synthesis of infectious bacteriophages in an e. coli-based cell-free expression system
Uploaded: 2017-08-17T15:00:00
Description: Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

这种材料基于工作支持的海军研究办公室奖号码 N00014-13-1-0074 （马路），人类前沿科学计划授予数量 RGP0037/2015年 （马路），和两国科学基金会授予 2014400 （马路）。

注意： 以下扩增步骤和提取方法在很大程度上可归纳为许多双链 DNA 噬菌体，例如， 噬菌体 T7、 肠杆菌噬菌体 T4 或肠杆菌噬菌体 λ (L)。它们主要用于噬菌体的基因组不是现成的商业来源购买。
 1.噬菌体扩增 注： 单斑块、 多周期 （次贷危机） 噬菌体生产技术是很好的描述为 T4 噬菌体在陈 等人 14 下面的噬菌体扩增和 DNA...

<ol>
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后陈等人的技术14的次贷危机，关键的一步被达到确定二重感染的适当条件时。最严密控制主机应变能力，能够承受二重感染的参数通常是感染噬菌体的初始浓度。宿主细胞必须在之前初次感染极少量的噬菌体对数生长阶段。最终，噬菌体也会达到对数增长，目标是以允许噬菌体数字迅速超越细胞计数，允许为每个宿主细胞噬菌体的多个副本。这迫使细胞进入准稳定状态?...

在过去的十年，无细胞表达技术工程化到地址小说在新兴的多学科研究领域相关的合成和定量生物学中的应用。最初用于表达蛋白独立的一个活的有机体，新无细胞 TXTL 系统已为这两个基础和应用科学1，2，大大扩大这一技术的范围。新一代的 TXTL 平台已被设计成用户友好，效率更高 （达 2 毫克/毫升的蛋白质合成的批处理模式3），更多功能转录4，一级和模块化，以方便地集成小说自然或扩展现有的生物系统5，6的合成功能。尤其是，无细胞 TXTL 系统已成为方便快速原型的遗传程序，例如调控元件或小基因电路7，8，9，通过减少设计-生成-测试循环到几天。引人注目的是，新的 TXTL 系统也是能够处理大型节目如体内大肠杆菌噬菌10，11，强展示足够的性能支持活跃基因组重组完成合成的 DNADNA 编码生活实体。
TXTL 系统目前相比传统体外建设性的生化分析中的很多技术优势。无细胞 TXTL 链接到最终的产品在减少和开放的环境中，而不是复杂的生活细胞的细胞质基因表达的过程。TXTL 利用 DNA 生化系统体外，重建现代 DNA 装配技术，是负担得起和快除了不需要讲究蛋白质纯化步骤。无细胞的表达提供了大部分的生化反应，从而使分子间的相互作用12深夹层中的组件直接访问。TXTL 反应，人可以改变意愿，几乎是不可能在一个活细胞的生物化学和生物物理设置。鉴于这些优势和最近有所改善，TXTL 技术变得越来越流行作为合成和定量生物学替代平台。虽然快速增长的研究团体使用 TXTL 和 TXTL 正成为标准技术在生物工程中的，至关重要的是要了解如何使用这种平台，发展与执行 TXTL 反应，以及相关的适当做法结果的解释。
在这篇文章中，我们描述了如何使用所有的大肠杆菌TXTL 系统在一锅反应合成噬菌体从其基因组11、 如 MS2 (RNA，3.4 kb)，ΦΧ174 (ssDNA，5.4 kb)，和 T7 (双链 Dna，40 kb)。我们展示如何噬菌体大量合成反应 （镁和钾浓度） 的生化设置的一些变化。分子的拥挤，通过 PEG 范围 8000 浓度，效仿戏剧性影响噬菌体合成了几个数量级。实现的这类大型的生化系统中单试管反应，同时概括的转录，翻译，过程和自组装，是有趣的处理有关的生物学和生物物理学的基本问题。10 (基因调控、 自组装)，以及为开发应用程序，如重新调整用途噬菌体的功能，打造新的纳米结构13。除了实用于 TXTL，我们通过空斑法测定提供噬菌体扩增，基因组提取和纯化，和噬菌体量化的方法。这手稿 》 中提出的方法是适当的人员使用大肠杆菌提取基于无细胞系统和感兴趣的噬菌体。
议定书 》 提出了这项工作可以概括如下： 1） 噬菌体扩增 (一天 1： 准备接种细胞，第 2 天： 单斑块、 多个噬菌体生长和浓度和第 3 天： 纯化噬菌体)，2) 双链基因组 DNA 提取（苯酚/氯仿萃取），和 3) 无细胞噬菌体反应和效价实验 (一天 1： 板宿主细胞并使琼脂平板，第 2 天： 无细胞反应和宿主细胞前文化，和一天 3： 宿主细胞文化和噬菌体效价)。

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		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:162pt;height:15.75pt" width="216">Ultracentrifugation tubes</td>
			<td class="xl21" style="width:168pt" width="224">Beckman Coulter</td>
			<td class="xl21" style="width:219pt" width="292">344057</td>
			<td class="xl20" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Conical tubes</td>
			<td class="xl22" style="width:168pt" width="224">Falcon</td>
			<td class="xl21">352070</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Gradient maker</td>
			<td class="xl21">BioComp Gradient Master</td>
			<td class="xl22" style="width:219pt" width="292">see Anal Biochem. 1985 Jul;148(1):254-9.</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Syringe and Blunt Cannula</td>
			<td class="xl21">Monoject</td>
			<td class="xl21">8881513918 and 888202017</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Wide-bore pipette tips</td>
			<td class="xl21">Fischerbrand</td>
			<td class="xl21">02-707-134</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Plaque counter</td>
			<td class="xl21">New Brunswik Scientific</td>
			<td class="xl21">Colony Counter Model C-110</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Culture tubes</td>
			<td class="xl22" style="width:168pt" width="224">Fischerbrand</td>
			<td class="xl21">14-961-33</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Cell-free system</td>
			<td class="xl22" style="width:168pt" width="224">Mycroarray Inc</td>
			<td class="xl22" style="width:219pt" width="292">Mytxtl</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">BioComp Gradient Master</td>
			<td class="xl17" style="width:168pt" width="224">BioComp Instruments</td>
			<td class="xl17" style="width:219pt" width="292">Model 105ME</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">LB agar plate recipe</td>
			<td class="xl20"></td>
			<td class="xl20"></td>
			<td class="xl24" style="width:512pt" width="683">25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td>
		</tr>
	</tbody>
</table>

synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

新一代的无细胞转录翻译 (TXTL) 系统，工程有更大的灵活性和模块化，提供新颖的功能来执行基础和应用科学中试管反应。在过去的十年，无细胞 TXTL 已成为广泛的新颖的多学科研究领域相关的定量和合成生物学强大技术。新的 TXTL 平台是特别有用的构建和审问生化系统通过合成或天然基因电路的执行。在体外TXTL 已被证明的高度概括，方便快速原型调控元件和生物网络，以及分子自组装机制发现生命系统中。在这篇文章中，我们描述如何传染性噬菌体，如 MS2 (RNA)，ΦΧ174 (ssDNA)，和 T7 (dsDNA)，完全由他们的基因组中使用所有的大肠埃希氏大肠杆菌、 无细胞 TXTL 系统的一锅反应合成。合成三个体内大肠杆菌噬菌被量化使用空斑法测定。我们展示如何合成噬菌体产量取决于反应的生化设置。分子的拥挤，通过控制浓度的 PEG 8000，效仿的订单的大小影响合成噬菌体的量。我们还描述如何放大噬菌体和如何净化它们的基因组。组协议和结果提出了这项工作应该是多学科研究人员感兴趣的在无细胞合成生物学与生物工程学院。

我们显示四个标志性的成果。在图 1中，我们提出一套的阴性对照，以确保无细胞 TXTL 系统和噬菌体 DNA 股票不沾染生活大肠杆菌细胞。我们验证无细胞 TXTL 系统是免费的完整大肠杆菌细胞由电镀无效的基因组 DNA （图 1A和图 1B） 两个非孵化和孵育反应液的污染。如?...

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

新一代的无细胞的转录翻译平台被设计在生化系统体外基因电路执行构建。在这篇文章中，我们描述如何从其基因组，使用所有的大肠杆菌细胞免费 TXTL 系统合成噬菌体 MS2、 ΦΧ174 和 T7，等。

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Research

JoVE Journal

Bioengineering

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

科研

生物工程

Synthesis of an Intein-mediated Artificial Protein Hydrogel

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

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

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

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 ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

快速生长的纳氏弧菌的无细胞蛋白表达

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

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

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 ...

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

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

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. ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

测量组织直肠炎和莱蒂克药物血栓分析功效的体外系统

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...