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><tbody><tr><td>LB Broth, Miller&amp;nbsp;</td><td>Fisher Scientific</td><td>12-795-027</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP9744500</td><td></td></tr><tr><td>Carbenicillin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP26481</td><td></td></tr><tr><td>M9, Minimimal Salts, 5x</td><td>Sigma-Aldrich</td><td>M6030</td><td></td></tr><tr><td>Casamino Acids&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1424-100</td><td></td></tr><tr><td>Magnesium Sulfate, Anhydrous</td><td>Fisher Scientific</td><td>M65-500</td><td></td></tr><tr><td>Calcium Chloride, Dihydrate</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Dextrose (D-Glucose), Anhydrous</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>NEB Turbo Cell Line</td><td>New England Biolabs</td><td>C2984l</td><td></td></tr><tr><td>Oligonucleotide Primers</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>25N synthesis, DSL purification</td></tr><tr><td>Q5 High-Fidelity Polymerase</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>Q5 Reaction Buffer</td><td>New England Biolabs</td><td>B9027S</td><td></td></tr><tr><td>dNTP Solution Mix</td><td>New England Biolabs</td><td>N0447S</td><td></td></tr><tr><td>Agarose</td><td>Bioexpress</td><td>E-3120-125</td><td></td></tr><tr><td>Ethidium Bromide, 1%</td><td>Fisher Scientific</td><td>BP1302-10</td><td></td></tr><tr><td>Gel Extraction Kits</td><td>Epoch Biolabs</td><td>2260250</td><td></td></tr><tr><td>GenCatch Plasmid DNA Miniprep Kit</td><td>Epoch Biolabs</td><td>2160250</td><td></td></tr><tr><td>AatII</td><td>New England Biolabs</td><td>R0117S</td><td></td></tr><tr><td>SacII</td><td>New England Biolabs</td><td>R0157S</td><td></td></tr><tr><td>HindIII-HF</td><td>New England Biolabs</td><td>R3104S</td><td></td></tr><tr><td>EcoRI-HF</td><td>New England Biolabs</td><td>R3101S</td><td></td></tr><tr><td>Cutsmart Buffer</td><td>New England Biolabs</td><td>B7204S</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>New England Biolabs</td><td>B0202S</td><td></td></tr><tr><td>ColiRolle Glass Plating Beads&amp;nbsp;</td><td>EMD Millipore</td><td>7101-3</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Fisher Scientific</td><td>BP1755-10</td><td></td></tr><tr><td>NHS-Desthiobiotin (DTB)</td><td>Thermo Fisher Scientific</td><td>16129</td><td></td></tr><tr><td>Succinimidyl Trans-4-(maleimidylmethyl) Cyclohexane-1-Carboxylate (SMCC)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S1534</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)&amp;nbsp;</td><td>Fisher Scientific</td><td>BP231-100</td><td></td></tr><tr><td>Succinimidyl 3-(2-pyridyldithio) Propionate (SPDP)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>S1531</td><td></td></tr><tr><td>NHS-LC-LC-biotin</td><td>Thermo Fisher Scientific</td><td>21343</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>31490</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS), 10x Solution</td><td>Fisher Scientific</td><td>BP399500</td><td></td></tr><tr><td>Streptavidin (SA)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>21145</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Ethylenediaminetetaacetic acid (EDTA)&amp;nbsp;</td><td>Fisher Scientific</td><td>S311-500</td><td></td></tr><tr><td>Tween 80&amp;nbsp;</td><td>Fisher Scientific</td><td>T164-500</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Scientific</td><td>H325-4</td><td></td></tr><tr><td>3, 3&#039;, 5, 5&#039;-tetramethylbenzidine (TMB)</td><td>Fisher Scientific</td><td>AC229280050</td><td></td></tr><tr><td>Vivaspin 500 Centrifugal Concentrators&amp;nbsp;</td><td>Viva Products</td><td>VS0192</td><td></td></tr><tr><td>Sodium Acetate, Anhydrous</td><td>Fisher Scientific</td><td>BP333-500</td><td></td></tr><tr><td>96-Well Polystyrene Plates</td><td>Thermo Fisher Scientific</td><td>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

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

科研

生物化学

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

遗传学

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

生物工程

<meta charset="utf8"></head><body>用于微区室化合成细胞的 CFPS 方案

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

<meta charset="utf8"></head><body>作者聚焦：优化 CFPS 系统以进行合成细胞构建

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

用于构建合成细胞的无细胞蛋白质合成系统

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

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

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

利用合成生物学工程师活细胞接口，具有可编程材料

摘要

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此视频中的章节

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Engineering Intracellular Protein Sensors in Mammalian Cells

模块化 DNA 设备的自动机器人液体处理组件

用于构建合成细胞的无细胞蛋白质合成系统

用于构建合成细胞的无细胞蛋白质合成系统

用于构建合成细胞的无细胞蛋白质合成系统

基于多步法链位移光刻的基因功能表面固定化研究

用于长期无细胞基因表达传导的多层微流体平台

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

使用DNA对细胞进行简单、经济、模块化的图案化