Name: engineering adherent bacteria by creating a single synthetic curli operon
Uploaded: 2012-11-16T12:00:00
Description: Watch this Scientific Journal Video about Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon at JoVE.com

我们感谢其他成员在里昂INSA-ENS iGEM比赛的球队（克莱门斯Gonthier，∙格夫雷梅拉妮·维维安·Chansavang，玛蒂尔德杜蒙德，亚历山大辰声，玛歌Jaulin，奥莉海牙，五鬼LY，托马斯Poinsot，绿柱石罗耶·贝特朗·朱莉Soula，迈克尔Vonzy ，皮埃尔·伊夫曾德尔，盖尔·奥利维尔Brette，Soufiane Bouhmadi，Chambonnier，劳拉伊扎尔，Aurianne Kroiss，菲利普勒琼，艾格尼丝罗德里格，阿尔诺Rondelet，西尔维Reverchon和Valerie加鼎银行），我们的赞助商的财政支持（生物梅里埃公司，法国电力公司，基金会，艾西斯腾INSA，ENS里昂INSA里昂生物科学部），F. Wisniewski律师染料批判性的阅读这份手稿和CC诗博士应变礼物。 B.合用的接收到博士奖学金从区罗纳 - 阿尔卑斯大区。

1。 Biobrick设计和合成的Curli子的<ol><li>确定的基因的组织和内源性的转录和转译信号的curli基因定位。这些信息被收集在专门的数据库（如RegulonDB 一个或EcoGene b）仔细阅读，并完成有关的出版物。数据管理和预分析，在硅片进行复制管理器软件 c。 </li><li>选择相关的编码序列。选择一组绝对必需的基因，为curli合成（csgB和csgA编码合成纤维单体）和出?...

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关键步骤合成生物学的方法，最关键的一步，这是基因设计。合成基因设计必须一丝不苟，以确保有效的系统生产。两个基因编码的纤维单体和三种基因编码的蛋白质参与在其分泌系统已组装与一个强大和金属诱导的启动子，以建立一个新的一种新的应用程序的功能单元，用于：核的污水的生物除污。按计划和预测，此设备导致增强的高curli生产，这是通过增加在培养基中的钴?...

逆境支持的细菌黏附在生物修复，生物催化或微生物燃料电池中起着重要的作用。生物修复过程使用的微生物降解有机物质的能力，或修改的金属分布（固定，挥发）或形态。这些有益的活动中观察到水生和陆地生态系统，而且在人工开发的系统，以处理工业和生活废弃物污染的水。微生物活性的强度和质量依赖于物理化学因素，但也对微生物的生活方式（自由漂浮或嵌入到生物膜）。生物膜的形成是相关联的新陈代谢，促进抗性的生物杀灭剂通过不同的机制。因此，应鼓励这种现象在大多数的生物修复过程。此外，工程大肠杆菌细胞控制生物膜的形成已被成功地应用到固定化全细胞传感器，生物芯片上2-3。 适应发生微生物高浓度的金属通过如吸附到细胞外基质成分的不同的机制，外排泵的激活或特定的运营商能够集中在金属进入细胞。在实验室规模，提高这些细菌的活动，通过基因工程使金属污染的有效和廉价的治疗，尤其是在薄弱数量的情况下，剧毒金属拉古等人所描述的2008年4月 。在这种情况下，细菌修复代表经典的化学过程，使用离子交换树脂相比具有竞争力和节约成本的方法。作者描述了一个E.钴的吸收和保留第一所淘汰的外排泵编码基因RCNA，然后通过改造一个多拷贝质粒使生产过剩的大肠杆菌底盘基因工程优惠钴的吸收转运。会出现这样的菌株作为一种有效的替代离子交换树脂处理放射性污水，但没有解决的一个关键问题是恢复受污染的细菌在结束的过程4。因此，我们的工作目标是设计一个自定义设计的应变能坚持逆境的支持，如玻璃或塑料。 当中的一整套确定的革兰氏细菌的粘附和粘附菌毛，我们选择设计系统，让curli生产的。 Curli薄（直径为2-5 nm）和高度聚集的淀粉样纤维突出的E.大肠杆菌和沙门氏菌表面作为一种非结晶性及不溶性基质5-7。 Curli也参与了非生物表面的殖民化和发展的生物膜8。 Curli最近汞离子结合9。被称为淀粉样蛋白确实具有较高的亲和性的金属离子，如Cu 2 +，Zn 2 +的和Fe 3 + 10。此属性可能会进一步提高去污的金属污染的废水。的CSG群集为生产的curli纤维负责两个趋异转录的操纵子（ 图1）的构成。 csgB，csgA和CSGC基因构成的意义操纵子，编码两个curli的亚基，CsgA和CsgB的。 CSGC似乎是内参与氧化还原活性和，影响CsgG孔隙行为11的curli合成系统。然而，由于缺乏CSGC在大多数curli生产菌表示的相应的蛋白质只提供一个secundary的控制curli生物合成水平。为了简化系统，我们一直在选择工作的最小数量的基因。 该csgDEFG在管理和运输operonencodes必需的蛋白质的细胞表面CsgA和CsgB。 CsgD是的csgBAC操纵子的转录激活和生物膜形成的控制通过控制生产curli菌毛和其它生物膜组分如纤维素12，并通过抑制鞭毛生产13中起着关键的作用。 CSGE，体育用品联合会和CsgG的构成curli特定分泌装置中的外 - 膜通过该主要curli亚基蛋白CsgA的是作为可溶性蛋白分泌。的聚合CsgA是依赖于与膜结合的成核剂：蛋白CsgB（评论14） 在体内 。复杂的调控途径已被证明是涉及多个双组分系统控制curli基因的表达15-16。这些复杂的法规允许的细菌，形成厚厚的生物膜的通过的curli的生产环境线索，但难以控制的工业应用。为了促进经济复苏的相遇人-酿一个工业过程中的细菌，细菌固定到固体支持物的确需要通过以下来控制和定义的参数（s）。粘附curli属性链接到它们的淀粉样蛋白性质17，并可以用来改善生物修复过程，但要创建一个简单的和容易控制的装置。 这7个基因18，其中一组的5个绝对需要为curli合成（csgB和csgA编码合成纤维单体）和出口（CSGE体育用品联合会和csgG的，编码的curli的分泌复杂）的基因构建合成的操纵子。为了摆脱“自然”的规定，curli，包括这5个CSG的强劲和钴overinducible的的启动子的控制下（ 图2）的基因，设计并合成了一种人工合成的操纵子。一步一步的分析curli编码区和同步功能的设计过程正题操纵子进行说明。可视化和量化细菌粘附聚苯乙烯和玻璃两种方法进行了说明。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> pIG2 </td><td></td><td></td><td> pUC57（ORI促进pMB1，2710 bp）的一个3165 bp的经EcoRI / PstI片段合成P RCN - csgBAEFG操纵放大器ŕ </td></tr><tr><td> pUC18中</td><td></td><td></td><td>多拷贝质粒（2686 bp的促进pMB1 ORI），放大器ŕ </td></tr><tr><td> S23 </td><td></td><td></td><td> SSC1（GFP标记的MG1655，礼品的CC士）/ pIG2 </td></tr><tr><td> S24 </td><td></td><td></td><td> SSC1/pUC18 </td></tr><tr><td> 氯化钴</td><td>西格玛</td><td></td><td> 0,1 M储备溶液在室温下保存</td></tr><tr><td> M63 </td><td></td><td></td><td> 29 </TD&gt; </tr><tr><td> 24 - 孔板</td><td> Nunc公司</td><td> 55429 </td><td>聚苯乙烯24孔板</td></tr></tbody></table>

engineering adherent bacteria by creating a single synthetic curli operon

这里描述的方法，包括重新设计E.一个强大和金属-overinducible的启动子的控制下，通过组装的最小数目curli基因的大肠杆菌的附着性，并在可视化和量化的细菌粘附的产生的收益。这种方法适用于相应的工程原则的抽象和标准化的合成生物学，和的结果中的BBa_K540000的Biobrick（最佳新Biobrick设备，设计，IGEM 2011）。 第一步包括在响应于金属中专门讨论curli生产过剩合成操纵子的设计，因此，在增加的粘附能力的野生型菌株。原curli操纵子进行了修改，在硅片，以优化转录和翻译信号和逃脱的“自然”规定的curli。这种方法允许，以测试成功我们目前了解的curli生产，。 MoreoveR，简化的curli切换的内源性的复杂子（超过10个转录调控因子识别）一个简单的金属调控启动子调控，坚持更容易控制。 所述第二步骤包括：通过简单的方法实施的粘附能力的定性和定量评估。这些方法适用于大范围的附着的细菌，无论参与生物膜形成的生物结构。粘附试验在24孔聚苯乙烯板，提供了一种快速的初步的细菌生物膜的结晶紫染色后的可视化。这种定性试验可以削尖的量化遵守百分比。这样的方法是非常简单的，但更精确的比如前所述1既具有良好的重复性和再现性的唯一的结晶紫染色。可视化GFP标记的细菌玻片上的荧光或激光CONFOCAL显微镜可以通过直接观察的现象，以加强与24孔板试验所获得的结果。

在硅片注释野生型CSG序列E.大肠杆菌 K12与转录和转译信号的优化已经允许设计的单一的合成curli操纵子P RCN-CSG示于图3（补充数据中所有序列）。协议2和协议3，坚持与curli生产用于可视化和量化。使用24孔聚苯乙烯板（协定书，2）的结晶紫染色生物膜的形成在每个孔中的底部可以迅速可视化和它与野生型菌株似乎是由紫色颜色强度的差异较少的附着性比工程...

Watch this Scientific Journal Video about Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon at JoVE.com

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

的合成操纵子编码既分泌装置和curli纤维的结构的单体的设计进行说明。这些淀粉样蛋白和粘附聚合物的生产过剩允许的，坚持一个可衡量的收益<em&gt; E.大肠杆菌</em&gt;底盘<sup&gt; 1</sup&gt;。简单的方法来可视化和量化坚持进行说明。

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Research

JoVE Journal

Bioengineering

Procedure for Lung Engineering

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

合成，多层，自激振荡的声带模型制作

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Synthetic Spider Silk Production on a Laboratory Scale

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...