作者非常感谢来自美国的科学研究空军办公室从获奖FA9550-13-1-0108支持。作者还从美国的海军研究办公室确认来自奖N00014-15-1-2502支持，从研究所的关键技术和应用科学在弗吉尼亚理工学院和州立大学和美国国家科学基金会研究生科研经费奖学金计划，奖号1607310。

1.媒体和文化准备<ol><li> 通过混合25g的LB粉末库存用1L去离子（DI）水和高压灭菌在121℃的溶液中20分钟以消毒准备LB培养基（LB）的介质。 <ol><li>为了制备LB平板，琼脂15克（1.5％）灭菌前添加到LB培养基</li><li>制备在去离子水中1000倍羧苄青霉素（CB）（50毫克/毫升）的储备溶液。 </li><li>如果制备，其包括用于抗性转化体的选择的抗生素的LB培养基，等到无菌LB介质的温度低于60℃，然后添加抗生素库存的1微升1毫升的LB每个介质。 </li></ol></li><li> 对于M9基本培养基（表1），准备以下的单独的储备溶液:5X M9盐（56.4克/升），1M 硫酸镁 ，1M的CaCl 2，20％的葡萄糖和2％无生物素酪蛋白氨基酸。 <ol><li>在高压釜中安全瓶子，结合20 5倍的M9毫升盐，200＃181; 1M 硫酸镁 ，DI水为1M的CaCl 2，2毫升20％的葡萄糖，1毫升2％的无生物素酪蛋白氨基酸，和76.8毫升10微升升。 </li><li>高压釜在步骤1.2.1在121℃20分钟制备的溶液。 </li></ol></li><li>孵育所有文化中，在37℃，以400rpm搅拌。孵化时间取决于实验和应变，但通常持续8至12小时。 </li></ol> 2.代生物素产大肠杆菌 （质粒pKE1-的lacI-bioB） 注:基因电路包含两个部分:一个lacI抑制子 ，通过一个P L推动，TETO-1启动子导致组成型表达由于不存在一个四面体阻遏蛋白，以及含有在P trc启动-2生物素的表达系统子，其后驱动bioB的表达的强的核糖体结合位点（RBS）。所有克隆是在商业，快速分裂大肠杆菌 STRA执行了，这一最终的构建物转化到大肠杆菌 MG1655WT进行测试。引物（ 表2）商购。 <ol><li> 隔离来自大肠杆菌基因组的bioB基因通过进行全细胞聚合酶链反应（PCR）: <ol><li>成长的大肠杆菌 MG 1655WT细胞过夜37℃。 </li><li>在0.2毫升的PCR管中，结合在步骤2.1.1用无菌DI水9微升制备的过夜培养物1微升。 </li><li>孵育管中在95℃在一个热循环5分钟，并立即将管转移到-80℃冷冻10分钟，以裂解细胞。这使基因组DNA以用作PCR的模板。 </li><li>解冻溶液并加入（ⅰ）各引物nBioB2-f1和nBioB2-R的2.5微升，（ⅱ）5 5×聚合酶缓冲液100μL，（ⅲ）0.25微升DNA聚合酶，（ⅳ）0.5微升dNTP混合物的，以及（v）6.75微升的去离子水的25微升的总反应体积（ 表4）。 </li><li>罢工（ 即甩尾）管的端混合的内容。接着，立即快速降速管（〜2秒），以确保样品在管的底部。 </li><li>放置管在PCR热循环，并使用表3中的3分钟的额外步骤在95℃下在协议的开始所描述的程序。 </li><li>确认成功PCR，使用凝胶电泳 - 在Tris碱，乙酸和EDTA缓冲液（TAE）（1.0在去离子水+溴化乙锭的1.2％琼脂糖）。 PCR产物应该是1070个碱基对（BP）长。 </li><li>使用根据制造商的说明书的商业试剂盒从凝胶从步骤2.1.7提取DNA片段。 </li></ol></li><li> 隔离在P TRC-2启动子和来自质粒pKDL071 13（詹姆斯·柯林斯在麻省理工学院实验室提供）的操纵子的lacI: <ol><li>含增长大肠杆菌细胞在pKDL071质粒过夜LB +的Cb在37℃。 </li><li>提取使用根据制造商的说明在商业小量制备试剂盒的细胞中的质粒DNA。该质粒将作为PCR模板。 </li><li>遵循步骤2.1.4 PCR方法。至2.1.8。并作以下修改: </li><li>在步骤2.2.2质粒提取替换裂解的细胞。 </li><li>使用的引物1-f和1-R为盒式的lacI提取2.5微升。或者，使用的引物nBioB1-F和nBioB1-R对于p TRC-2提取2.5微升。 </li><li>执行凝胶电泳（步骤2.1.7）确认PCR产物的的lacI盒和P trc启动-2启动子位点。它们应分别为1213 bp和109 bp的。 </li></ol></li><li>通过重叠延伸（SOE）PCR使用剪接构建含bioB匣P trc启动-2中，引物nBioB2-f2的合成的核糖体结合位点（RBS）和bioB </EM&gt;基因。该thermocyler PCR程序在表3中。 </li><li> 插入结构（P L，TETO-1 + 的lacI和P TRC-2 + bioB）到pKE1-MCS质粒载体13骨干消化载体和限制性内切酶插入（詹姆斯·柯林斯在麻省理工学院的实验室提供）: <ol><li>提取使用限制性酶和凝胶电泳基因。每个反应含有（ⅰ）5微升10×反应缓冲液，（ⅱ）限制酶1的1微升，（ⅲ）1微升限制性内切酶2的，（ⅳ）的DNA中的至少1微克，以及（v）去离子水以使终体积到50微升（ 表5）。用酶的AatII和EcoRI为的lacI卡带和用酶HindIII和SacⅡ位为bioB卡带消化。 </li><li>反应被组装后，很快在37℃孵育前涡旋它们并短暂离心（〜2个），1小时。 </li><li>中消化，根据标准方法进行电泳制备凝胶。 </li><li>结合了凝胶电泳6X样缓冲消化DNA。 </li><li>使用2日志梯验证所需构建体的位置，从凝胶上切的DNA片段，并使用根据制造商的说明商业凝胶提取试剂盒，从凝胶中提取DNA片段。 </li></ol></li><li>使用光谱量化DNA和计算量为0:1,1:1和3:插入物1的摩尔比使用等式1到载体: <img alt="式（1）" src="/files/ftp_upload/55300/equation1.jpg" /> 式（1） 另外，在式1中，M是插入到载体中的比率（0，1，或3）中，X i是插入DNA的量，沸点i是在碱基对插入物的长度，碱基v是矢量的长度在碱基对，且X v是向量（50毫微克）的量。 </li><li>通过组合混合连接反应（ⅰ）1微升10X连接酶缓冲液，（ⅱ）1微升的T4连接酶，（ⅲ）50纳克载体DNA，（四）具体到每个反应插入DNA，以及（v）去离子水的质量，以10μL的总反应体积（ 表6）。 </li><li>孵育在室温下（RT）1小时。 </li><li> 在步骤2.7结扎孵化，准备化学感受态细胞。 <ol><li>分装1毫升过夜培养到1.5毫升离心管中。 </li><li>离心在16,200 xg离心1分钟。 </li><li>倒出上清，悬浮颗粒在冷冻200微升（冰）100毫米氯化钙2。轻轻吹打悬浮颗粒。 不要旋涡。 </li><li>放置在冰上的微量离心管10分钟。 </li><li>离心机，除去上清液，重悬在100微升冷却的100mM 的 CaCl 2沉淀，并再次放置在冰上的管中。 </li><li>离心管中的最后的时间和悬浮颗粒在50微升的冷却的100mM的CaCl 2。 </li><li>地点冰管，并立即使用化学感受态细胞。 </li></ol></li><li>加入5μL连接的DNA，在步骤2.6和2.7编写，以感受态细胞，在步骤2.8编写的每管。 </li><li>由撞击侧（即轻弹）将管短暂地搅动该管，然后将管在冰上30分钟。 </li><li>热在42℃震荡管45秒，将管返回到冰上2分钟。 </li><li>吸管细胞上选择性LB琼脂+抗生素板和扩散用玻璃镀珠的细胞。 </li><li>过夜孵育所述板在37℃。 </li><li>第二天早晨，拾取从镶嵌正极板菌落（ 即 ，1:1和3:1的平板）。挑选3个菌落如果0:1负控板显示没有增长，或选择5-8菌落如果0:1板有一定的增长。使用拾取菌落来接种5ml的LB +抗生素，并在37℃下搅拌生长8小时。 </li><li>提取从细胞中使用minipr质粒DNAEP试剂盒，按照制造商的说明进行操作。执行使用限制酶的测试切口（以下在步骤2.4.1详述的相同程序）。 </li><li>通过比较从正确构建体预期的结果的消化的DNA片段的长度识别与成功的构建体的细胞。携带成功质粒构建培养物可通过等份培养混合用无菌过滤，40％甘油的溶液（在DI水中），并冷冻在-80℃下将混合物保留下来。 注:质粒变换成其他大肠杆菌菌株（ 即 ，MG1655WT）可以通过下列用于制备化学感受态细胞的上述步骤来完成。 </li></ol> 3.细胞特性:生长曲线和剂量反应<ol><li>生长工程菌在LB培养基中过夜与适当的抗生素。 </li><li> 对生长曲线，制备50mL的最小M9培养基的使用和不使用IPTG（从0.5米每秒滴答）和/或DTB（从500微克/毫升的库存）如下: <ol><li>准备0毫微克/毫升DTB（0微升股票）/ 0毫米IPTG（0微升的股票）。 </li><li>准备200毫微克/毫升DTB（股票200微升）/ 0毫米IPTG（0微升的股票）。 </li><li>准备0毫微克/毫升DTB（0微升股票）/ 0.5毫米IPTG（股票50μL）。 </li><li>准备200毫微克/毫升DTB（股票200微升）/ 0.5毫米IPTG（股票50μL）。 </li><li>用过夜培养物接种于1:在上述指定媒体100。 </li><li>在一个96孔板，等分试样培养200微升，一式三份，向每个孔中。 </li><li>测量的OD 600，每5分钟以上24小时，在板读取器连续摇动并孵育在37℃。 </li></ol></li><li>对于剂量响应研究中，取代在pKE1-的lacI-bioB与mCherry实时光学定量bioB基因（红色荧光蛋白）。 </li><li>添加不同的IPTG量为0.1至5mm以诱导表达在LB培养基中。 </li><li> 100:以1:1的稀释过夜培养物接种。 </li><li>测量荧光15小时每隔30分钟。 </li></ol> 4.改造的细胞和上清液诱导制备生物素生产<ol><li>在LB培养基中生长pKE1-的lacI-bioB在大肠杆菌 MG1655株过夜。 </li><li>补充的M9介质DTB范围从30至200毫微克/毫升。 </li><li>加入0.5毫米IPTG诱导生物素的合成。 </li><li>接种过夜培养补充，无生物素最小M9培养基以1:100稀释。 </li><li>生长，离心细胞24小时后，收集生物素富集的上清液。 </li><li>测量与控制的间接和直接控制功能化表面富含维生素H上清。使用上清到位步骤5.23和6.12，分别是生物素的样品。 </li></ol> 5.间接控制方案官能表面处理<ol><li> 日准备Ë以下解决方案。 <ol><li>制备SMCC溶液由20毫克/琥珀酰亚胺反式-4-（maleimidylmethyl）环己烷-1-羧酸酯中的溶液（SMCC）在二甲亚砜（DMSO）。 </li><li>制备的SPDP溶液由20毫克/琥珀酰亚胺基3-（2-吡啶基）丙酸酯（SPDP）在DMSO中的溶液。 </li><li>在DMSO中制备20毫克/毫升的LC-LC-生物素。 </li><li>在磷酸盐缓冲盐水（PBS）制备10毫克/毫升辣根过氧化物酶（HRP）。 </li><li>制备10毫克/毫升的链霉（SA）的在PBS中。 </li><li>制备在PBS中的10毫克/毫升牛血清白蛋白（BSA）。 </li><li>制备在去离子水中的100mM二硫苏糖醇（DTT）。 </li><li>制备在PBS中的5mM ethylenediaminetetaacetic乙酸（EDTA）。 </li><li>准备在PBS 0.5％的酪蛋白。 </li><li>在去离子水配制20％吐温80的股票。 </li><li>在PBS准备0.05％吐温80（20％股份）。 </li><li>准备在去离子水醋酸钠50毫米。 </li><li>在DMSO准备1％TMB。 </li><li>准备3％H 2 O 2我ñ去离子水。 </li><li>在去离子水中制备2 MH 2 SO 4。 </li></ol></li><li>添加SPDP液1.4微升至20微升SA的解决方案。 </li><li>从孵育5.2溶液在室温1.5小时，用铝箔包裹，以避免暴露在光线。这个步骤可以使SPDP交联剂通过形成吡啶激活SA的氨基结合到SA。 </li><li>从步骤5.3中添加滴滴涕溶液到该溶液中的2.4微升并在室温下孵育1小时。这允许吡啶-2-硫酮裂解，产生巯基活化的SA。 </li><li>加入7.5μLSMCC解决方案，以72微升HRP解决方案，并在室温下孵育1.5小时，用铝箔包裹，以避免暴露在光线。这导致由氨基结合的马来酰亚胺活化的辣根过氧化物酶。 </li><li>混合用200微升BSA溶液17μLLC-LC生物素溶液，在室温下孵育1.5小时，用铝箔包裹，以避免暴露在光线。此步骤允许生物素结合Ť经酰胺键ØBSA。 </li><li>从步骤5.4，5.5传输解决方案和5.6离心浓缩旋管中分离出来。 </li><li>对于含有SA-SPDP，从步骤5.4，离心在10000 XG直到管卷管达到100μL或16分钟。填充旋管500微升用PBS-EDTA。 <ol><li>重复步骤5.8五倍。在4℃保存的股票。 </li></ol></li><li> 对于含有HRP-SMCC，来自步骤5.5，离心机以10,000 xg离心直至体积达到25微升或16分钟的管。旋管填充到500微升用PBS。 <ol><li>重复步骤5.9五倍。在4℃保存的股票。 </li></ol></li><li> 对于BSA含有生物素，从步骤5.6，离心机以10,000 xg离心直至体积达到100微升或12分钟的管。旋管填充到500微升用PBS。 <ol><li>重复步骤5.10四倍。 </li><li>离心机在10,000 XG直到体积达到100μL或12分钟。加入100微升的PBS到管。在4℃保存的股票。 </li></ol></li><li> 在4℃添加25微升以10毫克/毫升的SA溶液并储存25微升的10毫克/毫升的HRP溶液过夜。这将导致该SA经由硫醚键成为共轭与HRP的。 <ol><li>准备1:用隔夜的解决方案，并存储在4℃的冰箱4可行的解决方案。在-20℃储存剩余的溶液。 </li></ol></li><li>准备两个解决方案，包括在PBS BSA生物素（或BSA）的，通过添加BSA生物素股票（或10μLBSA股票）10微升至PBS的490微升。 </li><li>从步骤5.12中添加100μL的该溶液到96孔聚苯乙烯板的各孔中。 </li><li>孵育在37℃的板1小时，包裹在箔。 </li><li> 用0.05％吐温80洗涤平板的孔中。 <ol><li>添加0.05％PBS-吐温80的200微升到孔中。在室温下孵育2分钟。倒出液体。 </li> &lt;李&gt;重复步骤5.15.1三次。 </li></ol></li><li>添加0.5％的酪蛋白溶液的200微升的每个中的96孔板的孔中。 </li><li>孵育在37℃的板1小时。 </li><li>重复步骤5.15洗井的三倍。 </li><li>通过混合12mL的0.5％的酪蛋白溶液用0.05％吐温80的7.5微升制备溶液。 </li><li>使用步骤5.19制备的溶液10000:稀释SA-HRP股票1。 </li><li>添加在步骤5.20制备一个96孔板的每个孔中的溶液的80微升。 </li><li>添加20微升制备的生物素的样品到聚苯乙烯板的各孔中。在4.6中制备的上清液可以用作在此步骤中的生物素的样品。 </li><li>孵育在37℃的板1小时。此步骤允许自由生物素之间的竞争性结合并固定BSA生物素对SA-HRP结合位点。 </li><li>重复步骤5.15洗井的三倍。 </li><li>混合50mM乙酸钠溶液，1％TMB溶液，并在1000 3％的H 2 O 2溶液:10:1的比例（总体积20ml）。 </li><li>从步骤5.25至每孔加入该溶液的200微升，并允许坐在RT 15分钟，用箔覆盖。 </li><li>加的2 MH 2 SO 4 50微升至各孔以终止反应。用酶标仪测量OD 450。 </li></ol> 6.直接控制方案官能表面处理<ol><li> 准备以下解决方案。 <ol><li>在DMSO中制备20毫克/毫升的LC-LC-生物素。 </li><li>制备10毫克/毫升的HRP在PBS中。 </li><li>在PBS准备0.17微克/毫升SA。 </li><li>准备在PBS 0.5％的酪蛋白。 </li><li>在去离子水配制20％吐温80的股票。 </li><li>在PBS准备0.05％吐温80（20％股份）。 </li><li>准备在去离子水醋酸钠50毫米。 </li><li>在DMSO准备1％TMB。 </li><li>在去离子水中制备3％的H 2 O 2。 </li><li>制备2 MH 2 SO 4 的 </sub&gt;在DI水中。 </li></ol></li><li>加入7.5微升LC-LC生物素至72微升HRP，在室温孵育1.5小时，用铝箔包裹，以避免暴露在光线。此步骤通过酰胺键将导致生物素缀合物的HRP。 </li><li> 旋管内来自步骤6.2的溶液转移到离心浓缩 <ol><li>以10,000 xg离心离心该溶液，直至体积达到100微升或12分钟。填充旋管500微升用PBS重复离心和PBS另外四次。在4℃保存的股票。 </li></ol></li><li>从6.1.3添加100μL的该SA溶液至每孔的96孔聚苯乙烯板内。 </li><li>孵育在37℃的板1小时，包裹在箔。 </li><li> 用0.05％吐温80洗涤平板的孔中。 <ol><li>添加0.05％200微升吐温80到孔中。在室温下孵育2分钟。倒出液体。 </li><li>重复6.6.1三次。</li></ol></li><li>添加0.5％的酪蛋白溶液的200微升的每个中的96孔板的孔中。 </li><li>孵育在37℃的板1小时。 </li><li>重复步骤6.6三次以洗涤井。 </li><li>使用步骤6.3准备的解决方案10000:稀释生物素HRP股票1。 </li><li>添加在步骤6.10制备一个96孔板的每个孔中的溶液的80微升。 </li><li>添加20微升制备的生物素的样品到聚苯乙烯板的各孔中。在4.6中制备的上清液可以用作在此步骤中的生物素的样品。 </li><li>孵育在37℃的板1小时。此步骤允许为固定SA结合位点自由生物素和生物素HRP之间的竞争性结合。 </li><li>重复步骤6.6以洗涤孔三次。 </li><li>混合50mM乙酸钠溶液，1％TMB溶液，和3％的H 2 O 2溶液以1000:10:1的比例（总体积20ml）。 </li><li>从步骤6添加溶液的200微升。15至每个孔，并在室温下孵育15分钟，用箔覆盖。 </li><li>加的2 MH 2 SO 4溶液50微升至各孔以终止反应。用酶标仪测量OD 450。 </li></ol>

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作者什么都没有透露。

我们已经提出用于连接工程活细胞与官能材料表面的新策略。这通过开发能够当用IPTG诱导合成生物素的水平升高的细胞系实现的。生物素的水平升高然后可用于修改功能化表面。该协议详细讲述了工程师大肠杆菌细胞系，以及如何创建两个不同的官能表面。 在这个协议中的关键步骤发生在整个工程细胞株的构建。以避免下游的问题，表征与两个生长曲线的工程细胞株?...

这里，我们报告开发能够从工程改造的细胞系响应于化学信号的可编程衬底的步骤。 1，我们通过创建生物素-链霉接口，响应生物素由合成工程化的大肠杆菌（大肠杆菌）细胞中产生的做到这一点。此前，可编程的表面经过特别设计，从毒素检测2点护理诊断3到国防和安全的广泛应用。 4虽然可编程表面可以是传感器和致动器是有用的，它们可以通过与以适应不同的环境挑战的能力赋予他们进行"更聪明"。相比之下，即使是简单的微生物，如大肠杆菌 ，具有内在的适应性，并能够应对成熟而往往出人意料解决方案的挑战。这种适应性使E.大肠杆菌群，通过其复杂的基因网络控制，经济有效地寻求资源，创建5产品附加值，6，甚至电源微观尺度的机器人。 7通过结合活细胞与使用可编程表面中的自适应优点，我们可以创建能够响应不同环境条件的一个聪明衬底。 合成生物学给了研究人员的新能力，生物体的行为进行编程。通过工程细胞包含新的基因调控网络，研究人员可以设计出多种编程行为细胞。 8，9之外的基础研究中，可使用的应用，如控制材料组件和生物学生产增值产品这些行为。 10在此，我们我们详细介绍如何利用合成生物学的工具来恩gineer 大肠杆菌菌株在诱导合成生物素。该菌株是通过使用限制性内切酶克隆的方法来组装一个质粒，pKE1-的lacI-bioB开发的。该质粒中，当转化到大肠杆菌 K-12菌株MG1655，赋予细胞以表达bioB，生物素合成的必需酶水平升高的能力。当转化的细胞用异丙β-D-1-硫代半乳糖苷（IPTG）诱导，并设置有生物素的前体，脱硫生物素（DTB），制作了生物素的水平升高。 生物素与链霉结合相互作用是在自然界中发现的最强的非共价键中的一个。这样，生物素 - 链霉相互作用既充分表征和生物技术高度使用。 11在这个手稿中，我们提出采用生物素-链霉亲和的互动感知和检测细胞生产生物素具有功能化表面两种策略。我们参照这些对立表面，"间接"和"直接"控制方案。在间接控制方案中，细胞产生的生物素与已经缀合和固定在聚苯乙烯表面为链霉结合位点的生物素竞争。此外，链霉抗缀合有辣根过氧化物酶（HRP）。 HRP修饰3，3&#39;，5，5&#39;-四甲基联苯胺（TMB），以产生光信号，12可以由在450nm（OD 450）定量分光吸收（ 即光密度）进行监测。因此，间接控制方案允许研究人员通过监测OD 450信号的attentuation测定细胞生产生物素。 直接控制方案利用直接固定到链霉亲和材料表面并允许细胞产生的生物素和生物素化HRP链霉结合位点的竞争链霉亲和素事件。再次，在细胞产生的生物素的相对水平是通过测量的OD 450信号监测。 两者合计，改造的细胞和官能化表面允许我们通过在活细胞中诱导的网络控制可编程表面的属性。换言之，我们已经创建了一个系统，该系统通过这些系统连接起来需要生物体的适应性和可靠性和说明书中的工程材料界面的优点。

<table border="0" cellpadding="0" cellspacing="0" width="1072">
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">LB Broth, Miller&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">12-795-027</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Agar</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP9744500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Carbenicillin&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP26481</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">M9, Minimimal Salts, 5X</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6030</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Casamino Acids&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP1424-100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Magnesium Sulfate, Anhydrous</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Calcium Chloride, Dihydrate</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">C79-500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dextrose (D-Glucose), Anhydrous</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">NEB Turbo Cell Line</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">C2984l</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Oligonucleotide Primers</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">N/A</td>
			<td>25N synthesis, DSL purification</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Q5 High-Fidelity Polymerase</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">M0491S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Q5 Reaction Buffer</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">B9027S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">dNTP Solution Mix</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">N0447S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Agarose</td>
			<td style="width:209px;">Bioexpress</td>
			<td style="width:119px;">E-3120-125</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Ethidium Bromide, 1%</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP1302-10</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Gel Extraction Kits</td>
			<td style="width:209px;">Epoch Biolabs</td>
			<td style="width:119px;">2260250</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">GenCatch Plasmid DNA Miniprep Kit</td>
			<td style="width:209px;">Epoch Biolabs</td>
			<td style="width:119px;">2160250</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">AatII</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">R0117S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">SacII</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">R0157S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">HindIII-HF</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">R3104S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">EcoRI-HF</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">R3101S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">Cutsmart Buffer</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">B7204S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">T4 DNA Ligase</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">T4 DNA Ligase Reaction Buffer</td>
			<td style="width:209px;">New England Biolabs</td>
			<td style="width:119px;">B0202S</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">ColiRolle Glass Plating Beads&#160;</td>
			<td style="width:209px;">EMD Millipore</td>
			<td style="width:119px;">7101-3</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Glycerol</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP229-1</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP1755-10</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:527px;">NHS-Desthiobiotin (DTB)</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16129</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Succinimidyl Trans-4-(maleimidylmethyl) Cyclohexane-1-Carboxylate (SMCC)&#160;</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">S1534</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dimethyl Sulfoxide (DMSO)&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Succinimidyl 3-(2-pyridyldithio) Propionate (SPDP)&#160;</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">S1531</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">NHS-LC-LC-biotin</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">21343</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Horseradish Peroxidase (HRP)&#160;</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">31490</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphate Buffered Saline (PBS), 10X Solution</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP399500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Streptavidin (SA)&#160;</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">21145</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP1600-100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dithiothreitol (DTT)</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP172-5</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Ethylenediaminetetaacetic acid (EDTA)&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">S311-500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Tween 80&#160;</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">T164-500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Hydrogen Peroxide</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">H325-4</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">3, 3&#39;, 5, 5&#39;-tetramethylbenzidine (TMB)</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">AC229280050</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Vivaspin 500 Centrifugal Concentrators&#160;</td>
			<td style="width:209px;">Viva Products</td>
			<td style="width:119px;">VS0192</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Sodium Acetate, Anhydrous</td>
			<td style="width:209px;">Fisher Scientific</td>
			<td style="width:119px;">BP333-500</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">96-Well Polystyrene Plates</td>
			<td style="width:209px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">266120</td>
			<td></td>
		</tr>
	</tbody>
</table>

using synthetic biology to engineer living cells that interface with programmable materials

我们已开发出一种非生物-生物界面，允许工程细胞以控制官能化表面的材料性质。 大肠杆菌细胞的合成工程化菌株和官能化材料的界面:该系统是通过创建两个模块制成。在本文中，我们详细介绍了基因工程利用分子克隆策略大肠杆菌菌株内选择行为的协议。一旦开发，当暴露于化学诱导该菌株产生的生物素的水平升高。此外，我们创建两个不同的官能化的表面，其中每一个能够以细胞合成的生物素响应详细协议。综上所述，我们提出了一个方法来创建一个允许工程细胞，以控制非生物基材料成分和组装链接，非生物，生物系统。

代表性的结果在附图五个数字呈现。首先，我们提出以图形克隆过程（ 图1），以便读者能够在视觉遵循用于创建合成工程化的大肠杆菌菌株的关键步骤。为了表征细胞的种群动态，我们提供由在人口为600nm（OD 600）测定的光密度所产生的生长曲线（ 图2）。然后，我们显示了调节基因网络是如何验证，通过使用mCherry作为bioB?...

Watch this Scientific Journal Video about Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials at JoVE.com

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

本文提出了一系列的开发改造的细胞和，使综合工程大肠杆菌控制和操纵可编程材料的表面功能化表面的协议。

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

using-synthetic-biology-to-engineer-living-cells-that-interface-with

Research

JoVE Journal

Bioengineering

8.8K 次观看.  Carnegie Mellon University. 本文提出了一系列的开发改造的细胞和，使综合工程大肠杆菌控制和操纵可编程材料的表面功能化表面的协议。 

视频: Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

密封飞升商会阵列的无细胞生物学

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

利用合成生物学工程师活细胞接口，具有可编程材料

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