作者要感谢大卫·蒂雷尔博士（加州理工学院）的盛情质粒的礼物pQE9交流10 ATRP 12，汤姆·缪尔博士（普林斯顿大学）的盛情质粒KanR表- IntRBS-NpuNC-CFN 11的礼物，松田武久博士（技术，白山市，石川，日本金泽大学）的盛情质粒PET30-CUTA -提示1 10，博士和杰伊·D·基斯林（加州大学伯克利）的盛情质粒的礼物pJD757 13的礼物。这项工作是由美国国家科学基金会职业，美国空军叶和诺曼·哈克曼高级研究计划中的一部分支持。

1。质粒的构建注意：所有的基因都在使用每制造商的规格的Phusion高保真DNA聚合酶的标准PCR反应扩增。用于克隆的引物已经预先9所述。所有结构都列在表1中 。 <ol><li>要生成CUTA-NpuN（N，表1）： <ol><li> PCR扩增来自质粒PET30-CUTA-提示1 10和KanR表-IntRBS-NpuNC-CFN 11，分别CUTA和NpuN基因，使用适当的引物。 </li><li>消化这些片段与适当的限制性内切酶，并依次将这些片段插入T7启动子和一个C-末端6xHistidine标签以产生N个（ 图2A）之间的pET26b载体中。 </li></ol></li><li>以产生NpuC-S-CUTA（C，表1）： <ol><li> PCR扩增NpuC，CUTA和S fragmen吨[AG 3（PEG）] 10从质粒KanR表-IntRBS-NpuNC-CFN 11，PET30-CUTA-提示1 10和pQE9 AC 10 ATRP 12，分别使用相应的引物。 </li><li>消化这些片段与适当的限制性内切酶，并依次将这些片段插入T7启动子和一个C-末端6xHistidine生成C（图2B）之间的pET26b载体中。 </li></ol></li><li>要生成NpuC-S-SH3 LIG-CUTA（C-SH3 LIG， 表1）： <ol><li> PCR扩增CUTA使用含SH3 的LIG（PPPALPPKRRR）和柔性接头（GGGGS）2，以产生片段SH3 LIG-CUTA引物。 </li><li>在C与片段SH3 LIG-CUTA更换CUTA基因。 </li></ol></li><li>以产生SH3-GFP（ 表1）： <ol><li>利用质粒pJD757 13放大SH3结构基因适当的引物。 </li><li>此融合片段的GFP基因并将其插入到T7启动子（ 图2C）和一个C-末端6xHistidine标签之间的pET26b载体中。 </li></ol></li></ol> 2。蛋白表达<ol><li>变换50μl的化学感受态大肠杆菌 BL21（DE3）中与适当的表达质粒。 </li><li>转化后，连续稀释这些细胞，并镀上他们LB培养基（LB）/含有50微克/毫升卡那霉素的琼脂平板上。 </li><li>含孵育转化细胞在37℃〜15小时板。 </li><li>培养后，挑包含50-100个菌落的平板，重悬的所有殖民地的5毫升LB培养液中。 </li><li>转让悬浮到1升的LB肉汤含有卡那霉素（50微克/毫升）和生长的细胞，在37℃下用250rpm振荡。监测吸光度在600nm（OD 600）。成长的文化，直到OD 600〜0.8。 <ol><li> C和C-SH3 LIG，诱导蛋白表达，加入异丙基β-D-1-硫代半乳糖苷（IPTG）诱导的培养物（1mM的终浓度）孵育的培养在37℃下搅拌4小时以250rpm一边摇动。 </li><li>对于N和SH3-GFP，冷却文化〜18℃下浸泡在培养瓶中在冰水浴〜5分钟。诱导蛋白表达，加入IPTG至文化（1mM的终浓度）和孵化培养在18℃，14〜18小时，在250转一阵晃动。 </li></ol></li><li>蛋白表达后，离心培养在6000×g离心20分钟，在4℃下，以收集沉淀。店铺细胞沉淀在-80℃直至使用。 </li></ol> 3。蛋白质纯化<ol><li> 氮净化（变性 ​​条件） <ol><li>以10毫升/克的湿颗粒重悬细胞沉淀物在缓冲液A（ 表2）。 </li><li> IMMERSe为丸粒悬浮液在冰水浴中，并通过超声处理裂解细胞（放大器10中，用1秒脉冲和6秒的暂停1分钟）。 </li><li>离心溶胞产物以16,000×g离心20分钟，在4℃下</li><li>弃上清。重新悬浮在缓冲液DA颗粒（含有8M尿素），并离心悬浮液以16,000 xg离心20分钟，在4℃下</li><li>通过5ml的镍 - 次氮基三乙酸（NTA）柱预先用缓冲液平衡过的DA传递上清液。 </li><li>用30毫升缓冲液的DA辅以45毫米咪唑洗柱。用20 ml缓冲液的DA辅以150 mM咪唑洗脱纯化蛋白。 </li><li>降低尿素浓度的蛋白样品&lt;1毫在由任一个在3.1.7.1或3.1.7.2给出的下列方法： <ol><li>在使用管与&lt;20 kDa的截止4℃过夜透析蛋白在DPBS缓冲液（ 表2）。 </li><li>离心纯化的蛋白质在30＆＃160; kDa的超滤离心柱于2800×g离心，4℃，直至体积小于1毫升加14毫升DPBS缓冲到列稀释的蛋白样品。重复离心/稀释步骤3次以上。 </li></ol></li><li>缓冲液置换后，加入二硫苏糖醇（DTT）对纯化的蛋白质（终2毫摩尔），并通过一个30 k​​Da的超滤离心柱于2800×g离心，4℃下离心浓缩蛋白到约100毫克/毫升</li><li>分装浓缩的蛋白质并储存于-80℃直至使用。 </li></ol></li><li>的C和C-SH3 LIG净化（自然条件） <ol><li>重悬细胞沉淀物在缓冲液B（pH 6.0）中（ 表2）以10毫升/克的湿颗粒补充有1X蛋白酶抑制剂混合物。用酸性缓冲液以最小化目标蛋白的蛋白水解降解。 </li><li>如3.1.2所述，超声破坏细胞悬液。离心LYS吃在16000×g离心20分钟，在4℃，并保持上清液。 </li><li>通过预先用缓冲液B平衡过的5毫升的Ni-NTA柱通过可溶性裂解物</li><li>用缓冲液B洗柱补充有45 mM咪唑，和洗脱目标蛋白在20毫升缓冲液B补充有150mM的咪唑。 </li><li>对于C，跳到步骤3.2.6。对于C-SH3 LIG，进行额外的离子交换纯化步骤，如步骤以消除部分降解的蛋白质3.2.5.1 3.2.5.3到<ol><li>降低C-SH3 LIG NaCl浓度&lt;1毫米以下的3.1.7中描述的过程。 </li><li>目标蛋白质加载到5毫升的阴离子交换珠状琼脂糖基质预先用磷酸钠缓冲液（50mM，pH值7.0）平衡柱。 </li><li>从由含10mM的Tris-HCl pH 8.0的buffe溶液运行梯度柱洗脱目标蛋白r以含有补充了1M NaCl的相同缓冲液中的溶液。取样品中蛋白质的洗脱和样品池与基于十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的最高纯度。 </li></ol></li><li>缓冲交换纯化的蛋白质转化为DPBS缓冲液中，如在3.1.7中描述。 </li><li>添加DTT至纯化的蛋白（终浓度2mM），将浓缩的蛋白质〜100毫克/毫升使用30kDa的超滤离心柱如在3.1.8中描述。分装和储存在-80°C，直到使用浓缩蛋白。 </li></ol></li><li> SH3-GFP的纯化<ol><li>用缓冲液A以10毫升/克湿重悬浮颗粒细胞沉淀。 </li><li>如在3.1.2描述的超声破坏颗粒的悬浮液。 </li><li>离心溶胞产物以16,000×g离心20分钟，在4℃，并收集上清液。 </li><li>通过预先用平衡有5毫升的Ni-NTA柱通过将上清液（可溶性裂解物）缓冲液A </li><li>用30毫升缓冲液A洗柱辅以45 mM咪唑。用20 mL缓冲液A的补充与150 mM咪唑洗脱纯化蛋白。 </li><li>缓冲液使用类似于在3.1.7中描述的方法交换纯化的蛋白质转化为DPBS缓冲液和浓缩的蛋白至〜150毫克/毫升使用30kDa的超滤离心柱如在3.1.8中描述。 </li><li>分装和储存纯化蛋白质在-80℃备用。 </li></ol></li><li>含CUTA纯化样品的SDS-PAGE分析<ol><li>稀释的每种纯化的蛋白在双蒸水，以减少氯化钠至〜1 mM的浓度。在此NaCl浓度，大多数CUTA三聚体蛋白质的运行如在SDS-PAGE凝胶的单体。 </li><li>混合样品与2×SDS样品缓冲液（0.5M Tris-盐酸，pH为6.8，20％甘油，10％w / v的SDS，0.1％w / v的溴酚蓝，2％β-巯基乙醇），孵育在95℃下5分钟。 </li><li>加载SAM普莱斯到12％SDS-PAGE凝胶。进行电泳，在200 V为〜50分钟的恒定电压。 </li><li>按照标准协议（ 图1C）染色用考马斯亮蓝R250观察蛋白质的凝胶。 </li></ol></li></ol> 4。水凝胶形成注：在本研究中制备的样品水凝胶含有1.6 mM的每个水凝胶构建块，除非另有说明。这种蛋白质浓度产生一个软而稳定的水凝胶。注意：将叠氮化钠（NaN 3的）被加入到水凝胶为0.5％w / v的终浓度，以防止细菌污染。 NaN 3的是剧毒的，必须特别小心处理，因为在材料安全数据表所示。 <ol><li>计算每个以实现在一个100μl的样品水凝胶为1.6mm的最终浓度所需的浓缩的蛋白质的量。例如： 氮浓度：100毫克/毫升 的N分子量：26.3 kDa的（ 见表1） 所需体积：100微升所需浓度：1.6毫米 <img alt="" fo:content-width="4.5in" fo:src="/files/ftp_upload/51202/book.JPG" src="/files/ftp_upload/51202/book.JPG" width="600" /></li><li>为了使100微升水凝胶（1.6毫米），混合C（X微升，按4.1计算量）用5％的NaN 3（10微升），按照100毫米DTT（5微升）和N（Y微升，体积计算4.1）2毫升的玻璃小瓶中。 </li><li>加DPBS缓冲液（（85 - X - Y）微升）到小瓶实现100μl的终体积，并通过使用吸管尖涡流运动手动混合所有成分。注意：溶液变成混合时非常粘稠。 </li><li> 2分钟离心混合物以8,000×g离心以除去气泡。 </li><li>孵育在室温混合物下放置过夜，以允许内含肽转拼反应达到完全。通过转动管倒置确认水凝胶的形成。该蛋白不会流动，如果形成的水凝胶。 </li><li>通过检查步骤4.2之前和在SDS-PAGE凝胶步骤4.5后收集的样品（各0.5微升），如在3.4（ 图1C）中所述判断的内含肽转拼收率。 </li></ol> 5。通过对接蛋白（DP）和扩展坞绿色荧光蛋白的固定化肽（DSP）互动<ol><li>为了使50微升GFP的官能水凝胶（1.2毫米），结合了C-SH3 LIG（倍微升，按照4.1计算）和SH3-GFP（Y微升，按4.1计算）在1.7摩尔比为1:1毫升微量离心管中，并在室温下搅拌30分钟，温育混合物中。 </li><li>添加5％的NaN 3（5微升），100毫米的DTT（2.5微升），（42.5 - X - &lt;EM&gt; Y）微升DPBS到同一管内。添加N（Y微升，按照4.1计算的）来实现的N和C-SH3 LIG摩尔比为1:1。通过使用移液管尖端由涡流运动混合样本。 </li><li>在8000×g离心2分钟，离心分离该混合物，在室温下孵育混合物过夜在黑暗中。水凝胶孵化期间封装SH3-GFP的形式。 </li></ol> 6。使用1.6毫米水凝胶作为固定支架在有机溶剂酶促反应<ol><li>使用HRP作为模型酶。制备HRP（28毫克/毫升或0.63毫摩尔）的DPBS中的贮备液。 </li><li>使30微升的水凝胶（1.6毫米）截留HRP，组合Ç内A（X微升，按照4.1计算）与HRP（2微升），5％的NaN 3（3微升）和DTT（1.5微升的100mM的） 1.7毫升离心管中。 </li><li>加入N（Y &lt;/ em&gt;的微升，按4.1计算），并DPBS（23.5 - X - Y）微升。混合用枪头带有旋转运动。 </li><li>在8000×g离心2分钟，离心反应混合物，并在室温下孵育过夜。 注意：用于以下活性测定的试剂有剧毒。通过相应的材料安全数据表使用特定的安全建议。 </li><li>对酶反应，淹没在水凝胶在1ml反应鸡尾酒的含N，N-二甲基- 对苯二胺（5.8毫米），苯酚（5.8毫摩尔）和叔丁基氢过氧化物（2.9毫摩尔）在正庚烷14用移液管尖，以增加水凝胶与溶剂接触的表面积手动破坏凝胶。 </li><li>检测HRP的产品，靛酚型染料，通过测量在不同时间在读板器（ 图5）取在546 nm的样品的光学吸收率。 </li> &lt;/ OL&gt;

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没有竞争的经济利益存在。

在这项工作中，我们展示了一个高度稳定的内含肽介导的蛋白水凝胶的合成。使用一个分裂内蛋白使得能够有条件地形成响应于两个液相成分混合的水凝胶。具体而言，分割内含肽共价连接两个液相积木通过转拼反应，得到交联单元，反过来自我组装成水凝胶两侧的多肽单元。混合诱导形成水凝胶的旁路中的单组分蛋白质凝胶，其中的色谱纯化柱的凝胶介导的堵塞可能发生的合成技术困难?...

蛋白质完全由水凝胶携带可能显著推进领域等不同的组织工程，药物输送和biofabrication 1。它们比传统的合成聚合物水凝胶的优点，包括生物相容性和非侵入性地支持生物活性的球状蛋白的结合的可能性。 在这项工作中，我们描述了通过拆分内含肽介导的蛋白转拼反应及其应用作为蛋白质固定支架（ 图1）形成一种新的蛋白质凝胶的发展。此水凝胶的积木两种蛋白嵌段共聚物各自包含一个分裂的内含肽（IN和IC）和多聚体交联蛋白质的亚单位的N-或C-末端片段。该DnaE内含肽自念珠藻punctiforme（NPU）用作分割内含肽2,3和一个小的三聚体蛋白（12 kDa）的来自激烈热 CUTA horikoshii &lt;/ em&gt;的用作交联剂蛋白4,5。不同的交联剂，通过内含肽催化的转拼反应结合，导致了高交联的蛋白网络（水凝胶）的形成。 NPU内含肽的选择，因为它的快速反应动力学（T 1/2 = 63秒）和高反式剪接率（接近80％）2,3。该CUTA蛋白被选为由于其高稳定性的交联剂。 CUTA三聚物具有接近150℃的变性温度，并保持三聚体四级结构中含有多达5个M盐酸胍4,6的解决方案。由于不同的交联剂亚基之间交换的物理水凝胶表面糜烂7的主要贡献者，在CUTA很强的亚基间相互作用应该阻止这种亚基的交流，从而导致更稳定的水凝胶。一这些积木还含有高度亲水的肽的S-片段作为中间嵌段，以促进水保留8。 两种水凝胶积木混合发起内含肽片段的IN和集成电路之间的转拼反应，生成一个较长的多肽链中与交联剂在两个终端。由多个这样的分子单元的交联剂彼此相互作用，从而形成高度交联的水凝胶网络（ 图1A）。一个具体的“基座肽”（DSP）掺入水凝胶的基石之一，以促进一个“对接蛋白”（DP）-标记的目标蛋白质为水凝胶的稳定固定。使用一个分裂内含肽介导的水凝胶组件不仅提供了用于蛋白质合成的水凝胶的额外的灵活性，但也可以使目标蛋白质的高密度，均匀的载荷在整个水凝胶，作为目标蛋白质前的水凝胶形成的装载。 内含肽介导的蛋白的水凝胶是高度站ble在水溶液中有小到没有3个月室温后检测的侵蚀。稳定性被保持在一个宽范围的pH（6-10）和温度（4-50℃），并在水凝胶也与有机溶剂相容。这样的水凝胶可用于双球状蛋白质的固定化：绿色荧光蛋白（GFP）和辣根过氧化物酶（HRP）。水凝胶包埋后者蛋白用于在有机溶剂中进行生物催化。

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			&#160;Ni Sepharose High Performance HisTrap column
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			GE Healthcare Life Sciences
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			HiTrap SP Sepharose FF ion exchange column
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			GE Healthcare Life Sciences
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</table>

synthesis of an intein-mediated artificial protein hydrogel

我们提出了一个高度稳定的蛋白水凝胶由一个分裂蛋白-蛋白催化的转拼反应介导的合成。这种水凝胶的组成部分有两个蛋白嵌段共聚物各自包含有作为拆分内含肽的交联剂，和一个半三聚体蛋白质的一个亚单位。一种高度亲水性的无规卷曲插入嵌段共聚物用于水保持量为1。两个蛋白的嵌段共聚物的混合触发一个内含肽转拼反应，产生具有交联剂的多肽单元在任一端，可以快速自组装成水凝胶。此水凝胶是非常稳定的酸性和碱性条件下，在温度高达50℃，并在有机溶剂中。剪切诱导破裂后的水凝胶迅速改革。成立一个“基座肽”到水凝胶积木使“对接蛋白”标记的靶蛋白的方便结合。所述水凝胶是用组织培养的生长培养基兼容，支持的20kDa的分子的扩散，并且使生物活性的球状蛋白质的固定化。内含肽介导的蛋白水凝胶作为有机溶剂相容的生物催化剂的应用证明了包封的辣根过氧化物酶和确证其活性。

示意图为内含肽介导的蛋白质凝胶的形成是在图1A。水凝胶的基石是蛋白质共聚物CUTA-NpuN（N）和NpuC-S-CUTA（C）（ 图1A，表1）。 NpuN / C是自然分割DnaE的N-/C-fragments内含肽从发菜punctiforme（NPU）。 CUTA是来自激烈horikoshii 4,5稳定的三聚体蛋白质。纯化的N和C中的还原剂DTT存在下的混合导致形成三分之一蛋白-连接产物（j：CUTA-S-CUTA）（ 图1A和1C）...

Watch this Scientific Journal Video about Synthesis of an Intein-mediated Artificial Protein Hydrogel at JoVE.com

Synthesis of an Intein-mediated Artificial Protein Hydrogel

我们提出了一个分裂内含肽介导的蛋白水凝胶的合成。这种水凝胶的组成部分是各含有作为拆分内含肽的交联剂，和一个半三聚体蛋白质的一个亚单位2蛋白共聚物。两个蛋白共聚物的混合触发一个内含肽转拼反应，产生的多肽单元，其自组装成水凝胶。此水凝胶是高度的pH和温度稳定，与有机溶剂相容的，并且很容易地集成功能的球状蛋白。

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

synthesis-of-an-intein-mediated-artificial-protein-hydrogel

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JoVE Journal

Bioengineering

12.0K 次观看.  Texas A&M University, College Station. 我们提出了一个分裂内含肽介导的蛋白水凝胶的合成。这种水凝胶的组成部分是各含有作为拆分内含肽的交联剂，和一个半三聚体蛋白质的一个亚单位2蛋白共聚物。两个蛋白共聚物的混合触发一个内含肽转拼反应，产生的多肽单元，其自组装成水凝胶。此水凝胶是高度的pH和温度稳定，与有机溶剂相容的，并且很容易地集成功能的球状蛋白。 

视频: Synthesis of an Intein-mediated Artificial Protein Hydrogel

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

科研

生物工程

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

一种简便快速制备磁响应元件的水凝胶基微型机械的添加剂制造技术

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

三维印刷多孔纤维素纳米复合水凝胶脚手架

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

以自动化和标准化方式制造基于水凝胶的3D培养模型的开源技术平台

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.