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）和出口（CSGE体育用品联合会和csgG的编码的柯林分泌复杂的），构建合成的操纵子。从FASTA格式中的数据的基础上，提取所选择的序列。 ，由于csgGFE集群是在大肠杆菌中的反义大肠杆菌基因组，将它的反向的补充counterp的这个序列使用Clone管理工具操作过程分子反转分子的艺术。粘贴csgEFG背后csgBA序列使用的功能"结扎"（克隆结扎）。 </li><li>添加适当的启动。通过放置在五个选定curli基因的控制下，启动子P RCN，curli预测被过度生产的钴和镍的存在下。 RCN的基因编码外排泵，镍和钴的解毒（RCNA）和其同源的金属调节器（rcnR）负责。 RcnR控制RCNA和以响应于Ni和Co 19其自身基因的表达。的RCN的序列包括rcnR CDS，整个RCN间区域加41第一核苷酸RCNA:placedin，前面的csgBAEFG嵌合序列（ 图1，提供的整个构造的序列作为补充数据）。 </li><li>优化的转录信号。一个完美的核糖体结合位点（或完美的RBS = AAGGAGGTATATA）中的csgBA DNA序列中的第一个ATG前加入。第二完美的RBS中添加了前面的csgEFG序列。内源性RBS的csgB和体育用品联合会和csgG基因是保守的。 </li><li>为了适应iGEM比赛的标准，设备必须被夹击一个的标准BioBrick前缀和后缀， 生态 RI，含有限制性酶切位点的Pst I（前缀）和SPE我和 Xba I（后缀）。粘贴对应的序列，在每个端部的装置 d。 </li><li>消除任何生态RI，PST我，SPE I 和 Xba I识别位点的设备。为了便于进一步组装过程中，BioBrick部分本身可能不包含任何这些限制性酶切位点。电子限制分析设备显示PstⅠ位点在csgA序列，PstⅠ位点在rcnR序列和一个生态 RI网站的CSGE基因。这些站点分别位于位置80（MUT1），1340（MUT2）和1830年（Mut3）的设备序列（补充资料），并修改为沉默突变。 CTGCAG改变CGTCTG MUT1 PstⅠ位点 CTGCAG改变CAGCAG MUT2 PstⅠ位点 mut3 Eco RI位点GAATTC改变GAATT </li><li>通过翻译模拟运行的复制管理器软件（操作&gt;过程分子&gt;翻译分子）。使用序列比对程序BLAST验证之间的的野生型curli蛋白和合成操纵子所编码的蛋白质的同源性的完美。 </li><li>订购的合成操纵子。来自世界各地的许多公司提供商业基因合成服务，我们的的的伙伴是Genecust（卢森堡）。 4微克的人工操纵子（3165 bp）的几个星期后，已收到插入到pUC57（pIG2）。 </li></ol>2。可视化和量化粘附的细菌对聚苯乙烯<ol><li> M63基本培养基（葡萄糖0.2％）用2毫升的填充的24孔聚苯乙烯板的各孔中，并接种每孔106个细胞的过夜培养。生长的细菌在30°C为18〜48小时，无晃动。 （4孔）表示每一列的一种方式。在需要的时候添加适量的钴（25至100毫米）和抗生素（氨苄青霉素100μg.L-1）。 3的第一行是用来量化粘附的细菌通过平均3次重复的24孔板，剩下的最后一行是用于可视化的生物膜。 </li><li> 为3的第一行 :为每个孔的24孔板中，恢复包含浮游细胞的上清液。 </li><li>仔细冲洗每个孔用1ml了M63和游泳池的1毫升的洗涤与初始上清液中得到2.2）。该池被称为的游泳细胞（S）。 </li><li>在1毫升邻恢复生物膜f M63刮上下吹打（= B）。涡15秒。估价表面连接和游泳细菌的数目的光密度在600 nm（OD600），得到对应于每一种模式的粘附百分率。 </li><li>遵守的百分比是通过使用下面的公式计算:Bx100 /（3XS + B）。 </li><li> 只有最后一排的24孔培养板:放弃浮游细胞。 </li><li> M63的生物膜的板的底部上已经发展用1ml冲洗。 </li><li>干燥1小时的开闭板在80℃下</li><li>加入100μl20％的结晶紫在每个其次是广泛的可视化与水清洗附着的细菌，持续2分钟。 三个独立的实验（板）被执行，以确保可重复性。 </li></ol> 3。可视化在玻璃上粘附的细菌在显微镜下观察<ol><li>获取的荧光机箱。 ，E.大肠杆菌 SCC1Ë应变constitutivelŶ表达绿色荧光蛋白（GFP）从亲本菌株MG1655 20是一个合适的宿主质粒轴承的合成curli操纵子与没有可观察到的差异。 </li><li>变换SCC1 pIG2质粒（=合成curli操纵子插入的EcoRⅠ/ PstⅠ位点的pUC57质粒），以获得S23中的应变。变换SCC1与对照质粒（pUC18中），得到的对照菌株S24 21。 </li><li>生长过夜预培养的S23和的控制（S24）的菌株在30℃在M63培养基中与葡萄糖（0.2％）和氨苄青霉素（100μg.L-1）。 </li><li>接种在培养皿中加入100μl的预培养相同的培养基中的15毫升。添加适当浓度的钴（ 即 25μM）和阴性对照组无钴不要忘了。介绍3个矩形盖玻片，在每个培养皿。在30°C孵育过夜，无晃动。 </li><li>取出盖玻片来回在培养皿中，并仔细地排出。小心的下表面用一个小棉用70℃的乙醇浸渍的东西，每一个细菌的残留物擦干净。对于共聚焦观察，上清洁两个上端几毫米到允许进一步固定盖玻片。 </li><li>附着的细菌可直接可视化，但很快在荧光显微镜下，但仔细避免干燥的样品。 </li><li>共聚焦观察，在载玻片上的上表面覆盖的放置面朝上的生物膜沉积盖玻片。其形成的生物膜，再覆盖更大范围的盖玻片上，其被固定到载玻片清漆。反转设置，使生物膜将现在的下表面上。 </li><li>将设置下的激光共聚焦显微镜。右键Axioplan2 LSM510（蔡司）激光共聚焦显微镜被用来，在Platim平台f。 </li><li>设置。在488 nm激发GFP，并收集细菌荧光在500至600纳米的范围内<html> 。使用40倍油浸物镜，用于获取图像的激光扫描共焦模式。扫描的整体三维结构的从固体表面上形成生物膜，与生长培养基的接口，采用的步骤为1μm。 </li><li>执行立体的预测与IMARIS软件（位面，苏黎世，瑞士）。确定生物膜厚度通过分析沿统计的纵向部分的平均的z值。量化生物膜biovolumes的共聚焦Z-栈中提取所描述的其他地方22。 </li></ol> 一个 RegulonDB提供机械的操纵子的组织，它们的分解有关的转录单位，启动子和其Σ型，基因和它们的核糖体结合位点，终止子，具体的转录调节因子的结合位点，以及其组织成监管短语。ET ="_blank"&gt; http://regulondb.cs.purdue.edu/index.jsp B的EcoGene数据库包含更新信息的E.大肠杆菌 K-12基因组和蛋白质组序列，其中包括广泛的基因书目。的主要EcoGene焦点已经重新评估翻译起始位点。 <a href="http://ecogene.org/" target="_blank">http://ecogene.org/</a> Ç <a href="http://www.scied.com/dl_cmp9d.htm" target="_blank">http://www.scied.com/dl_cmp9d.htm</a> ð <a href="http://partsregistry.org/wiki/index.php?title=Help:BioBrick_Prefix_and_Suffix" target="_blank">http://partsregistry.org/wiki/index.php?title=Help:BioBrick_Prefix_and_Suffix</a> Ë春洲诗，南洋理工大学，新加坡的礼物。 的f Platim微观平台UMS3444生物科技杰兰- Lyon Sud酒店。

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Online. 8, 194-215 (2006).</li><li>Chavant, P., Gaillard-Martinie, B., Talon, R., Hebraud, M., Bernardi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+device+for+rapid+evaluation+of+biofilm+formation+potential+by+bacteria.">A new device for rapid evaluation of biofilm formation potential by bacteria.</a> J. Microbiol. Methods. 68, 605-612 (2007).</li><li>Miller, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experiments in molecular genetics. , (1972).</li></ol>

没有利益冲突的声明。

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量增强。的适当的转录信号在所选择的启动子（P RCNA），和高效率的转译信号的存在的这样的成功的结果附加在前面的每一组curli基因（csgBA和csgEFG）（参见图3）。此外，当使用多拷贝质粒，注意<html>要支付的拷贝数（S）的启动子控制的调节。在这里，所用的启动子（P RCNA）的主要调节RcnR多拷贝提供了从相同的质粒（ 图3）。 的限制，可能修改为了确保一个完美的reproductibility，坚持一直进行测试，在同一品牌的聚苯板（见表）。荧光显微镜与荧光标记的菌株是比较容易的，但这一要求可以通过使用荧光染料，如祥发，或质粒携带的紫胶 -GFP融合23克服。非生物如聚苯乙烯和玻璃表面的细菌附着取决于离子强度或渗透压的培养基中。获得最好的结果通常在基本培养基或稀释后的LB 22，24。 在掌握这种技术未来的应用领域或方向内容"&gt;这里所描述的方法来量化细菌粘附是快速和廉价的，然而，大量的菌株或培养条件的特征，和/或以获得详细的结构表征生物膜，基于激光扫描共聚焦显微镜的高通量方法使用96孔微量滴定板结合，必须考虑到25。 铁大桥局的筛选系统，原卡尔加里生物膜的移动设备26也可以被使用。此系统是基于微量滴定板，用一个可移动的盖子，有96栓允许水表超声波发生器（通过超声处理对中断后的显微镜观察生物膜结构27，或定量细胞生物膜基质的使用铁大桥局高通量（ HTP）的含量，Innovotech，埃德蒙顿，加拿大）。另一个系统，生物膜环测试，监视如何包含在培养基中的惰性的顺磁性珠的形成过程中被固定的生物膜28，并且可以用于量化生物膜的形成。中铁大桥局和生物膜环测试需要特定的材料，更昂贵的比在这项研究中所使用的基本消耗品。 相对于现有的方法的技术意义要创建一个单一的，独立的curli操纵，我们尝试了两种方法并行:这里描述的一种综合方法，一个典型的涉及诱变的方法，以消除内部生态 RI 和 Pst I酶切位点，PCR步骤的结扎。两种方法都在相同的时间开始，但经典方法通过以下方式获得的操纵子的序列分析表明不希望的突变。因此，合成的设备，更高效，更快速，比传统的克隆和突变的程序。

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菌株所示（ 图4A）。定性的方法是加强了定量测量，显示的比例，坚持高1.5倍，在工程菌（ 图4B）。此外，定量精度的<html> TIVE方法允许测量一个在存在的钴的浓度增加粘附显着增强。事实上，在媒体与钴的浓度为0μM，25μM或50μM，贴壁细胞的百分比达到25％，30％和40％（ 图4B）。用显微镜GFP标记的细菌生长在载玻片上，坚持能力，也可以进行比较。落射荧光显微镜观察（在黑色背景上的绿色细菌）表明工程菌株形成了几层视为生物膜，而野生型菌株只形成微菌落（ 图5）的大骨料。共聚焦显微镜分析提供的生物膜三维结构的整体图，和，确认工程菌是更粘附，形成更致密的和更厚的生物膜（ 图6）。 4176fig1.jpg"ALT ="图1"/&gt; 图1。 curli，野生型系统。curli由两种单体由csgB和csgA基因编码的。由于他们的信号肽，CsgA CsgB curli单体易位跨胞质膜通过Sec系统。 CSGE，体育用品联合会和CsgG的3个主要部分组成，即一个特定的机械组成的允许横跨外膜易位curli单体。 <img src="/files/ftp_upload/4176/4176fig2.jpg" alt="图2"/> 图2。一个强大和钴诱导型启动子（BBa_K540001）的启动子RCNA控制的转录抑制RcnR。该基因的转录从rcnR其内生性启动子rcnR，从P RCNA发散。存在的rcnR基因的阻遏副本相对于P RCNA监管确保正确的比例地区。在不存在的钴，RcnR结合到DNA上的RcnR框和防止充分的下游基因的转录激活。如果钴升高细胞内的浓度，钴的RcnR蛋白的结合防止固定阻遏的启动子的转录活性的PrcnA增加19。 <img src="/files/ftp_upload/4176/4176fig3.jpg" alt="图3"/> 图3。的合成curli操纵 。 curli基因被放置的控 ​​制下的P RCNA。 "rcnR从它自己的启动子基因的表达应提供足够的阻遏物控制钴依赖性curli基因表达。为了避免因curl​​i的单体的生产过剩（CsgB和CsgA），组件的特定的curli的分泌装置（CSGE，体育用品联合会和CsgG）的周质塞车有要生产过剩太。添加traductional信号是在<html> dicated在灰色（RBS =完美RBS）。 E = 生态 RI X = XBA I S = SPH I P = 的Pst I。这种合成的部分为Bba_K540000，使工程菌成为附着于玻璃，沙子或塑料通过curli生产过剩。 <img src="/files/ftp_upload/4176/4176fig4.jpg" alt="图4"/> 图4。粘附的细菌在24孔聚苯乙烯板的可视化和量化。 A.结晶紫染色生物膜形成由粘合聚苯乙烯上的细菌。B.聚苯乙烯与各种浓度的钴的野生型和改造的菌株粘附百分率。统计处理之间的差异，表明与小写字母（方差分析和Fisher最显着的差异测试，P &lt;0.05）。 76/4176fig5.jpg"ALT ="图5"/&gt; 图5。在黑色背景上附着的GFP标记的细菌在载玻片上。细菌是绿色的荧光显微镜图像 。箭头指向菌落。 <img src="/files/ftp_upload/4176/4176fig6.jpg" alt="图6"/> 图6。共聚焦microscropy协助在载玻片上的生物膜结构的三维重建:A.顶视图重建B.侧视图重建。

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 ...

engineering-adherent-bacteria-creating-single-synthetic-curli

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.4K 次观看.  Université de Lyon. 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 E.大肠杆菌底盘 1。简单的方法来可视化和量化坚持进行说明。

视频: Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

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

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

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

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 material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

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

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

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 ...

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

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

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

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

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

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 model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

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

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 increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

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

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

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

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

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 ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

通过创建一个的合成Curli的操纵子工程粘附的细菌

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