在这个视频文章进行的实验，开发了国际基因机器设计竞赛9。想感谢iGEM比赛的团队成员，卢克Bergwerff，彼得TM面包车Boheemen，Jelmer克诺森，乌戈·F.奎托·罗哈斯和拉蒙·Van Der Valk酒店协助这项研究。我们感谢韩Winde，斯特凡去角和Esengül的的耶尔德勒姆的有益讨论和主持这项研究。这项工作是支持的代尔夫特理工大学生物技术系，生物信息学实验室的代尔夫特，代尔夫特理工大学的Bionanoscience，油砂领导倡议（OSLI），梭哈studentenuitzendbureau，荷兰基因组计划，Kluyver中心，荷兰Biotechnologische Vereniging（STICHTING生物技术荷兰） ，DSM，香港传艺，格雷纳生物和杰能科。

1。 BioBrick大会<ol><li> BioBricks标准生物部件登记处提供的iGEM比赛的总部。构造一个新的BioBrick从现有BioBricks，与酶EcoRI和SpeI消化的的捐助BioBrick（高达1.0微克），用于定位的给体部分的受体部分的下游。用XbaⅠ和PstⅠ月刊用于定位给体部分上游的受体部分。添加第三个适当的限制性内切酶切割的捐赠者中的骨干。在用适当的缓冲液的总体积为20-25毫升，根据供应商（最终浓度：1倍）下进行消化。使用5个单位/微克DNA的限制性内切酶。 </li><li>消化受体BioBrick与EcoRI和XbaI或SpeⅠ和PstⅠ位。 </li><li>孵育消化（至少）一个小时，在37°C。通过热灭活的限制性内切核酸酶，温育在80℃下10分钟，并离心不久。 </li><li>结扎消化BioBrick部分（供体和受体）。由于XbaI和SpeI位产生兼容的DNA末端，创建混合站点不能被任何限制性内切酶切割产生一个新的“组合拳”BioBrick，两侧的4个标准的限制性酶切位点。在连接混合物的最终DNA浓度优选为〜100纳克/微升。执行用T4连接缓冲液（最终浓度：1倍）和T4连接酶（1单位/微克DNA）的总体积为10-15毫升的连接反应。 </li><li>连接混合物在16℃孵育至少3小时。 </li><li>大约一半的连接混合物进行转化反应。 </li><li>确认BioBrick与测序是正确的。 </li><li>要建立一个新的BioBrick合成的DNA，修改合成的基因在大肠杆菌中的优化表达大肠杆菌通过工具（ <a href="http://www.jcat.de/" target="_blank">http://www.jcat.de/</a> ）的JCAT网站。按照要求的B中，进行进一步的修改<html> ioBrick标准。这种标准化意味着每个BioBrick是之前由一个前缀和后跟一个后缀的利息的DNA序列组成。前缀和后缀的序列包含预定义的酶切位点，在剩下的质粒序列所没有的。这些标准的限制性位点，使人们有可能互换和弹性延长BioBrick插入9。前缀和后缀有下列序列： </li></ol><table border="1"><tbody><tr><td> 名称 </td><td> 序列 </td><td> 评论 </td></tr><tr><td>字首</td><td> 5&#39;GAATTCGCGGCCGCTTCTAG3“ </td><td></td></tr><tr><td></td><td> 5&#39;GAATTCGCGGCCGCTTCTAGAG 3&#39; </td><td>如果下面的部分是一个编码序列或任何部分开始“ATG” </td></tr><tr><td>后缀</td><td> 5&#39;TACTAGTAGCGGCCGCTGCAG 3&#39; </td><td></td></tr></tbody></table>ove_title“&gt; 2。烷烃转化静息细胞含量， 在体内 Fujii 等人 （2004）所记载的方法的基础上，进行该测定。 <ol><li>文化E.大肠杆菌表达烷烃羟化酶的系统（ <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a> ）和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的5毫升LB培养基中，与适当的抗生素过夜。 </li><li>转移到50毫升的新鲜LB（与抗生素）和500微升过夜培养孵育直到细胞浊度达到在600nm处的OD（光密度）为0.3。 </li><li>在4,000 rpm下离心10分钟，将沉淀重悬于5毫升0.1M的磷酸盐缓冲液（pH7.4）。 </li><li>于4,000 rpm离心10分钟，再次离心，将沉淀重悬于在5ml 0.1M磷酸缓冲液现在包含E2的盐和0.66％v / v甘油（氮缺陷T培养基）。 </li><li>测细胞的浊度（OD 600）。 </li><li>准备6毫升的细胞混合物的等分试样在25毫升封闭帽的玻璃烧瓶中并没有细胞对照组（E2盐+ 0.66％v / v甘油）。 </li><li>向每个烧瓶中加入100 nmol的烷烃。 </li><li>孵育24小时缩短率较高时，获得的混合物在37°C。 </li><li>测量外径600后孵化。 </li><li>提取物在培养基中的烃由乙酸乙酯中，用气相色谱法（见协议3），并确定在细胞培养中的烃浓度。 </li><li>除以转换每个烧瓶中（OD 600转换到干重）的总生物量中存在的链烷烃的总量，计算每单位的生物量的退化。实验的持续时间将结果除以每单位生物量，每单位时间产生的降解活性。平均取自三个单独的运行。 </li></ol> 3。烷烃转化酶活性检测， 在体外基本上按照由Li 等人 （2008年）记载的方法进行该测定。 <ol><li>文化E.大肠杆菌细胞表达：LADA基因（ <a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的在50毫升LB培养基中过夜与适当的抗生素。 </li><li> 500微升过夜培养转移到50毫升的新鲜LB（与抗生素），并孵育，直到细胞浊度达到在600nm处的光密度为0.6。 </li><li>在4,000 rpm下（4℃）离心10分钟，将沉淀重悬于5毫升的50mM Tris缓冲液中。 </li><li>超声处理的细胞（细胞破碎，LA生物系统公司）在40％占空比的输出控制4，保持解决方案的整个持续时间上冰。 </li><li>离心所得到的混合物于4,000 rpm离心5分钟，在4及d例如，C，以除去细胞碎片。将上清液转移到一个新的小瓶。 </li><li>确定通过Bradford测定法的细胞提取物中的总蛋白浓度。注意：使用玻璃小瓶中，以防止增加的背景和/或损失的蛋白质。 </li><li>准备的100毫升的混合物，含有0.1％（体积/体积）的烷烃和50mM Tris-HCl缓冲。 </li><li>加热该混合物，在100℃下5分钟。注意：执行此步骤仅适用于中高沸点的长链烷烃。 （ 例如：C 16：287°C）。 </li><li>为了达到最佳的烷烃中的溶解度，超声处理1分钟，同时仍然温暖，直到得到均匀的，粘稠的混合物。 </li><li>加入1mM的FMN的NADH，1mM的，1 mM的MgSO 4和0.01体积/体积的Triton X-100。 </li><li>准备6毫升等分封闭帽的在25毫升烧瓶。 </li><li>添加足够量的细胞提取物（取决于Bradford分析，最终浓度为5毫克蛋白/升）。准备一个没有控制蛋白质。 </li><li>孵育在60°C（最佳Ënzymatic活动）为24小时。 </li><li>提取物在培养基中的烃由乙酸乙酯中，用气相色谱法（见协议3），并确定在细胞培养中的烃浓度。 </li><li>计算每单位的生物量除以烷烃的总量由总蛋白质转换退化加入到每个烧瓶中（通过Bradford校准曲线）。进一步除以实验的持续时间，产生每单位每单位时间的细胞蛋白的降解活性。平均取自三个单独的运行。 </li></ol> 4。醋酸乙酯油气开采和浓度测量<ol><li>从水溶液中萃取链烷烃的加入2.5毫升乙酸乙酯（非极性solvant）到6毫升实验溶液。内部标准被添加到溶剂中，在浓度为0.1％（体积/体积）。的标准（ 例如，环癸烷）的变化取决于景点的峰的预期范围。 </li><li>可选：添加的TritonX-100的含水混合物，并在4,000 rpm下的10分钟离心处理，以使样品，以获得适当的双相系统。 </li><li>振荡，持续5秒（1,500 rpm下）和在室温下孵育，直到混合物的两相分开。 </li><li>删除最大量的有机层（顶部）和干燥的溶剂中，用无水硫酸镁干燥。 </li><li>删除用MgSO 4通过过滤（0.2微米），并转移到气相色谱仪小瓶滤液进行测量，或存储于-20℃下</li><li>确定的浓度用气相色谱法，使用CP-SIL 5CB柱（长度= 5米）。以分流模式注入10μl的样品（1:10，230°C）。设置列的气体流量，以1毫升/分钟（氦）。下面的烘箱温度程序是用来： <table border="1"><tbody><tr><td> 率 </td><td> 温度[°C] </td><td> 时间[min] </td></tr><tr><td> 0 </td><td> 50 </td><td> 7.5 </td></tr><tr><td> 50 </td><td> 90 </td><td> 1.0 </td></tr><tr><td> 50 </td><td> 110 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 130 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 145 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 160 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 170 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 185 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 210 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 250 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 320 </td><td> 2.0 </td></tr></tbody></table></li><li>峰进行积分并纠正的浓度相对于内部标准。 </li></ol> 5。醇/醛脱氢酶活性测定基本上按照由Kato 等人描述的方法进行该测定。（2010年）。 <ol><li>文化E.大肠杆菌细胞表达的的ADH（ <a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a> ）或乙醛脱氢酶 （ <a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a> ）基因和细胞携带一个空的的向量（ <a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a> ）的在50毫升LB培养基中，与适当的抗生素过夜。 </li><li> 500微升过夜培养转移到50毫升的新鲜LB（与抗生素），并孵育，直到细胞浊度达到在600nm处的光密度为0.6。 </li><li>在4℃下以4,000 rpm离心10分钟，将沉淀重悬于50mM Tris缓冲液的5毫升。 </li><li>与4的输出控制（保持该溶液在冰上超声处理过程中），超声处理的细胞溶液在40％的占空比。 </li><li>在4,000 rpm下离心5分钟，将所得混合物在4℃下以除去细胞碎片。 </li><li>确定的TAL蛋白的细胞提取物的浓度由Bradford法（注：使用的玻璃小瓶，以防止蛋白结合）。 </li><li> 96孔板中，用1​​80微升57 mM甘氨酸缓冲液含有NAD（终浓度1mM，pH 9.5）中的各孔中装入。 </li><li>加入5μl的醇（醛）进行测试，以井。注：热的长链醇的测定开始前，有一个液体的。对于每个醇（醛）基板的控制而不应该被添加（阴性对照）。此外，含有无细胞提取物的混合物的缓冲液和基板制备空白。 </li><li>预热板读板器（Tecan公司麦哲伦v7.0的）为15分钟，在37℃至允许系统的平衡。 </li><li>添加足够量的细胞提取物（取决于Bradford分析，最终浓度为5毫克蛋白/升）。准备一个没有控制蛋白质。 </li><li>测量的NADH的生产用分光光度计在波长340nm的每2-3分钟1小时，在37℃下&lt;/ P&gt; <li>从OD（340 nm）的斜率计算的NADH的生产速率。考虑到光路长度和6220 M -1 cm -1的消光系数的NADH。的NADH的生产速率除以蛋白质的总量添加到表达脱氢酶反应的细胞提取物中的活性（U /毫克全细胞蛋白）。计算从三个独立运行的平均值和标准偏差。 </li></ol> 6。 pCaiF表征<ol><li>文化E.大肠杆菌细胞表达pCaiF-GFP的结构（ <a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a> ）和细胞的启动子（ <a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a> ）在5毫升LB培养基过夜适当的抗生素隔夜。 </li><li>接种用50μl的过夜培养物中含有10克/升葡萄糖和抗生素5毫升M9，和成长过夜。 </li&gt; <li>继代培养的50微升的通宵生长细胞浊度达到到5毫升新鲜的M9 with10克/升并孵育，直到在600nm处的光密度为0.2。 </li><li>装入的96孔板用100μl的新鲜的M9培养基中，含有所需量的碳源，在各孔中进行测试。执行一式三份实验进行统计评价，添加相应的阴性对照（野生型大肠杆菌 K12）。 </li><li>添加5μL过夜培养介质中井。 </li><li>测量的生长曲线（OD 600）和GFP荧光（485 nm激发和520排放）使用读板器每10分钟18小时，在37℃下恒定摇动。 </li><li>计算的增长速度，从各自的测量的特定GFP内容的控制比较。计算至少三个独立实验的平均值和标准偏差。 </li></ol> 7。公差含量<ol><li>文化大肠杆菌表达PhPFDα和β基因（ <a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a> ）的隔夜在5毫升LB培养基和适当的抗生素。E.大肠杆菌表达： 拉达基因（ <a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）作为阴性对照。 </li><li>继代培养的10μl的产物到新鲜的5毫升LB（与抗生素）并孵育过夜，直到细胞浊度达到的光学密度为0.3至0.4，在600 nm。 </li><li>用新鲜培养基稀释培养，直到OD 600达到0.1。 </li><li>装入用180微升含有适当的抗生素和适当的最终浓度的有毒化合物（ 例如，0，4，8，10％的 n-己烷），一式三份的M9培养基中，在96孔板上。由于烷烃-水混合溶剂可能会导致两相系统，它是必不可少的，盘上的适当的控制（ 如d，增添菌株和各自的空白实验）。 </li><li>加入20μl的培养物（对照）入井。 </li><li>测量生物量浓度（OD 600）使用读板器每10分钟24小时在37℃下以恒定摇动。 </li><li>从各自的不同剂浓度的测量计算的生长率和阴性对照进行比较。计算的平均值和标准偏差至少三名独立运行的。 </li></ol> 8。同源互动映射<ol><li> STRING数据库（免费供教学使用）20上运行PostgreSQL服务器的应用他进行蛋白查询。的同源性互动地图应用程序标识使用STRING数据库在原有的宿主生物体相互作用的蛋白。用于在BLAST搜索找到同源基因中的靶生物的各相互作用的基因的序列。 Cytoscape的4 </s向上&gt;是用于可视化的结果的映射。他的软件工具可以下载： <a href="https://github.com/jcnossen/InteractionHomologMapping" target="_blank">https://github.com/jcnossen/InteractionHomologMapping。</a> </li><li>要进行映射，（1）输入BioBrick的ID和应用程序将自动下载标准生物零件，或（2）输入（粘贴）序列数据在应用程序从注册表中的部分序列数据。 </li><li>使用STRING数据库网站输入的氨基酸序列的找到STRING蛋白质的ID。 </li><li>使用BLAST确定一种蛋白质，具有较高的同源性。随后，应用程序列出了每个已知的相互作用蛋白在宿主生物中的同源物（ 如大肠杆菌 ）在源生物和搜索。 </li><li>假定的互动列表导出到文本或Cytoscape的。 </li></ol>

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Bacteriol. 178, 1248-1257 (1995).</li><li>Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+and+proteome+of+long-chain+alkane+degrading+Geobacillus+thermodenitrificans+NG80-2+isolated+from+a+deep-subsurface+oil+reservoir.">Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (13), 5602-5607 (2007).</li><li>Fujii, T., Narikawa, T., Takeda, K., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotransformation+of+various+alkanes+using+the+Escherichia+coli+expressing+an+alkane+hydroxylase+system+from+Gordonia+sp.+TF6.">Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6.</a> Biosci. Biotechnol. Biochem. 68 (10), 2171-2177 (2004).</li><li>Kato, T., Miyanaga, A., Haruki, M., Imanaka, T., Morikawa, M., Gene Kanaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cloning+of+an+alcohol+dehydrogenase+from+thermophilic+alkane-degrading+Bacillus+thermoleovorans+B23.">cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23.</a> J. Biosci. Bioeng. 91 (1), 100-102 (2001).</li><li>Kato, T., Miyanaga, A., Kanaya, S., Morikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+cloning+and+characterization+of+an+aldehyde+dehydrogenase+from+long-chain+alkane-degrading+Geobacillus+thermoleovorans+B23.">Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23.</a> Extremophiles. 14, 33-39 (2010).</li><li>Kieboom, J., De Bont, J. a. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial Stress Responses. , (2000).</li><li>Kotte, O., Zaugg, J. B., Heinemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adaptation+through+distributed+sensing+of+metabolic+fluxes.">Bacterial adaptation through distributed sensing of metabolic fluxes.</a> Mol Syst Biol. 6, 355 (2010).</li><li>Kremling, A., Bettenbrock, K., Gilles, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+global+control+of+Escherichia+coli+carbohydrate+uptake.">Analysis of global control of Escherichia coli carbohydrate uptake.</a> BMC Syst. Biol. 1, (2007).</li><li>Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., Rao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+long-chain+alkane+monooxygenase+(LadA)+in+complex+with+coenzyme+FMN:+unveiling+the+long-chain+alkane+hydroxylase.">Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase.</a> J. Mol. Biol. 376 (2), 453-465 (2008).</li><li>Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S. O., Neubauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+maximum+specific+uptake+capacities+for+glucose+and+oxygen+in+glucose-limited+fed-batch+cultivations+of+Escherichia+coli.">Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli.</a> Biotechnol. Bioeng. 73 (5), 347-357 (2001).</li><li>Okochi, M., Kanie, K., Kurimoto, M., Yohda, M., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+prefoldin+from+the+hyperthermophilic+archaeum+Pyrococcus+horikoshii+OT3+endowed+Escherichia+coli+with+organic+solvent+tolerance.">Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance.</a> Appl. Microbiol. Biotechnol. 79 (3), 443-449 (2008).</li><li>Rehm, H. J., Reiff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+occurrence+of+microbial+oxidation+of+long-chain+alkanes.">Mechanisms and occurrence of microbial oxidation of long-chain alkanes.</a> Adv. Biochem. Eng. / Biotechnol. 19, 175-215 (1981).</li><li>Rojo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+alkanes+by+bacteria.">Degradation of alkanes by bacteria.</a> Environ. Microbiol. 11 (10), 2477-2490 (2009).</li><li>Van Beilen, J. B., Panke, S., Lucchini, S., Franchini, A. G., Rothlisberger, M., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Pseudomonas+putida+alkane-degradation+gene+clusters+and+flanking+insertion+sequences:+evolution+and+regulation+of+the+alk+genes.">Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.</a> Microbiology. 147, 1621-1630 (2001).</li><li>Wang, L., Tang, Y., Wang, S., Liu, R. L., Liu, M. Z., Zhang, Y., Liang, F. L., Feng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+novel+thermophilic+Bacillus+strain+degrading+long-chain+n-alkanes.">Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes.</a> Extremophiles. 10 (4), 347-356 (2006).</li></ol>

没有利益冲突的声明。

BioBrick原则被用于构造的底架的与链烷烃的降解和该工具包为单个元件的一个原则的证明得到。几个实验提出在体内和体外活性烷烃降解途径酶的测量。所提出的工作成功地演示了一些方法，这些方法可以被用来确定酶的活性和表达在宿主有机体E.大肠杆菌合适的BioBricks后实施。此外，它是示出，该BioBrick原则可以用来设计一个有机体表达蛋白质所需的链烷烃的降解，提供​​了一个...

Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities 3. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil 1. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process water in order to reduce the need for freshwater uptake is needed. To facilitate the bitumen extraction and to ensure that downstream sites meet water quality guidelines for the protection of aquatic ecosystems, process water treatments are rapidly evolving 3.
To treat pollution of organic compounds, bioremediation technologies employing microorganisms are presently encouraged 1. Alkanes are the most abundant family of hydrocarbons in crude oil, containing 5 to 40 carbon atoms per molecule 7, 21. Many bacteria are known to degrade alkanes of various lengths via sequential oxidation of the terminal methyl group forming first alcohols, then aldehydes and finally fatty acids 8. Within this iGEM project several enzymes from different organisms were expressed and characterized, and made available via the BioBrick standard and Registry of Standard Biological Parts.
The well-studied alkane hydroxylase system of Gordonia sp. TF6 facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes using a minimum of four components: alkB2 (alkane 1-monooxygenase), rubA3, rubA4 (two rubredoxins) and RubB (rubredoxin reductase) 8, 21. Oxidation of long-chain alkanes (ranging from C15 up to C36) is reported to be performed by ladA, a flavoprotein alkane monooxygenase from Geobacillus thermodinitrificans NG-80-2 7, 15, 18, 22. LadA forms a catalytic complex with flavin mononucleotide (FMN) that utilizes atomic oxygen for oxidation. This results in the conversion of alkanes into the corresponding primary alkanol. The alcohols are further oxidized by alcohol and aldehyde dehydrogenases to fatty acids, which readily enter the &beta;-oxidation pathway 7, 21. A zinc-independent alcohol dehydrogenase from the thermophillic bacterium Geobacillus thermoleovorans B23 oxidizes medium-chain alkanols into their respective alkanals, using NAD+ as a cofactor 10. Aldehyde dehydrogenase from the same bacterium is able to catalyze the NAD+-dependent final step in the medium-chain oxidation 11.
In order to reduce induction costs and to maintain optimal proliferation of the bacterial system, the promoter pCaiF from E.coli was characterized. This promoter can regulate expression of the hydrocarbon degradation pathway components, and is regulated by cAMP-Crp levels, which in turn depend on glucose levels 6. At high extracellular glucose concentrations in the environment the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) level was low through the inhibition of adenylyl cyclase as a side effect of PTS mediated glucose transport. Conversely, during limitation (low glucose concentrations) the cAMP level increased and Crp bound to cAMP forming the complex, cAMP-Crp, which bound pCaiF and activated transcription of the downstream components 6, 14.
Wildtype E. coli can only tolerate moderate concentrations of hydrocarbons. To complete the toolkit, tolerance to hydrocarbons had to be addressed. Several organic solvent-tolerant bacteria are known to survive in water-solvent two-phase systems 12. Molecular components known to increase tolerance are chaperones that facilitate the correct folding of proteins. The prefoldin system from Pyrococcus horikoshii OT3, consisting of the proteins phPFD&alpha; and phPFD&beta;, was shown to increase hydrocarbon-tolerance 17.
The alkane conversion toolkit was constructed following the BioBrick principle, which is documented at the Registry of Standard Biological Parts 9. BioBricks are plasmids containing a specific functional insert that is flanked by 4 predefined restriction sites. The BioBrick inserts can be extended flexibly, allowing the construction of biological systems with new functions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>E. coli K12</td>
			<td>New England Biolabs</td>
			<td>C2523H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Octane</td>
			<td>Fluka</td>
			<td>74822</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexadecane</td>
			<td>Fluka</td>
			<td>52209</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>octanol-1</td>
			<td>Fluka</td>
			<td>95446</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dodecanol-1</td>
			<td>Sigma-Aldrich</td>
			<td>126799</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FMN</td>
			<td>Sigma</td>
			<td>F2253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>J.T. Baker Casno</td>
			<td>7487 889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell disrupter</td>
			<td>LA Biosystems</td>
			<td>CD-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Amersham pharmacia</td>
			<td>spec 2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>Magellan v7.0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Innova, 44</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BioBrick K398014: 
			BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4- BBa_J61100-rubB</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a></td>
			<td>Alkane Hydroxylase System 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398027: BBa_R0040-BBa_B0034-ladA</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398027" target="_blank">BBa_K398027</a></td>
			<td>ladA Protein Generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398018: BBa_J23100-BBa_J61101-ADH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a></td>
			<td>ADH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398030: BBa_R0040-BBa_B0034-ALDH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a></td>
			<td>ALDH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398326: pCaiF</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a></td>
			<td>pCaiF promoter 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398331: pCaiF-BBa_B0032-BBa_I13401</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a></td>
			<td>pCaiF measurement device 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398406: BBa_J23002-BBa_J61107-phPFD&alpha;-BBa_J61107-</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a></td>
			<td>Solvent tolerance cluster 
			Resistance: Chloramphenicol</td>
		</tr>
	</tbody>
</table>

a toolkit to enable hydrocarbon conversion in aqueous environments

这项工作提出了一个工具包，使烷烃转化大肠杆菌 ，并提出证明其适用的原则。该工具包包括多个标准的可互换的部分（BioBricks）的9解决烷烃转化，调节基因的表达和生存有毒碳氢化合物丰富的环境。 E.实施三个步骤的烷烃降解途径大肠杆菌介质和长链烷烃的转化率，以使各自的链烷醇，烷醛和最终链烷酸。后者则是通过本机的β-氧化途径代谢。为了方便的中链烷烃的（C5-C13）和环烷烃（C5-C8），4个基因（alkB2，rubA3 rubA4和擦除 ）的烷烃羟化酶系统从大头属的氧化。 TF6 8,21转化到大肠杆菌大肠杆菌 。为转换的长链烷烃（C15-C36）， 芽孢杆菌热拉达基因的实施。对于需要采取进一步措施的退化过程，ADH和ALDH（G.热 ）的10,11。活性测定采用静息细胞检测。对于每个氧化工序中，酶的活性进行了观察。 为了优化流程的效率，表达诱导低血糖条件下基板调节的启动，pCaiF，。 pCaiF是在E.大肠杆菌 K12和调节非葡萄糖碳源的降解中所涉及的基因的表达。 使用的工具包-针对生存的最后一部分-溶剂耐受的基因，PhPFDα和β，无论是从激烈热horikoshii OT3。有机溶剂可以诱导细胞应激和下降的生存能力产生负面affectin克蛋白质折叠。作为分子伴侣，PhPFDα和β改善蛋白质的折叠过程中， 例如，链烷烃的存在下。所示的一种改进的烃类公差在培养基中的增加的生长率（高达70％）10％n-己烷的存在导致这些基因的表达进行了观察。 总之，结果表明，该工具包能够E.大肠杆菌转换和容忍在水环境中的碳氢化合物。因此，它代表了一个可持续发展的解决方案，油整治利用合成生物学的方法的第一步。

Alkane conversion
The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. 
For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_...

Watch this Scientific Journal Video about A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments at JoVE.com

在水环境中实现碳氢化合物转化的工具包生物工程|视频

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

一个可持续发展的自动调节修复石油污染的细菌系统的设计采用标准的可互换的DNA部分（BioBricks）。一个工程<em&gt; E.大肠杆菌</em&gt;菌株用于在有毒的水溶液环境中，通过β-氧化降解烷烃。来自不同物种的酶显示烷烃的降解活性。此外，增加的耐受性<em&gt; N</em&gt;己烷，通过引入从烷烃耐受菌的基因来实现。

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

a-toolkit-to-enable-hydrocarbon-conversion-in-aqueous-environments

Education

Research

JoVE Core

JoVE Journal

Organic Chemistry

Biology

Bioengineering

Ecology

Unit 1

Ecosystems

Alkanes and Cycloalkanes

SCIENTISTS IN ACTION

14.0K 次观看.  Delft University of Technology. 一个可持续发展的自动调节修复石油污染的细菌系统的设计采用标准的可互换的DNA部分（BioBricks）。一个工程 E.大肠杆菌菌株用于在有毒的水溶液环境中，通过β-氧化降解烷烃。来自不同物种的酶显示烷烃的降解活性。此外，增加的耐受性 N己烷，通过引入从烷烃耐受菌的基因来实现。

视频: A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

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 ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

光图案蛋白质和细胞中的水环境中使用二氧化钛<sub&gt; 2</sub&gt;光催化

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

用作酶反应器的水滴及其电磁驱动

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

热、压水固化重组蜘蛛丝的材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...