作者感谢支持合成模型开发的国家耳聋与其他交流障碍研究所的资助R03DC8200，R01DC9616和R01DC5788。

制造序列（ 见图1）由声带模型层的模具，连续铸造硅层，并安装测试模型。该模型有四个不同的层次：身体，韧带，浅固有层和上皮细胞，除了单根光纤。后盾层添加声带模式，以方便个别层的准确位置。该模型的几何参数的定义见图 。 2，表1给出当前模型的参数值。在下面的章节，不同的有机硅混合比例指定为不同的层，这些生产材料的特性类似人类的声带组织（见表2）在小应变制度13。 1。模具制造和准备<ol><li>创建实体模型三声带层：SUPErficial固有层，韧带，和身体层。这通常是通过创建所需的几何形状的三维计算机辅助设计（CAD）模型，导出CAD模型作为立体光刻（STL）文件，和STL文件快速原型发送到定制机店。 </li><li>创建一个箱形的模具使用亚克力材质的薄件的形式。近似​​尺寸（并不重要），高2.54厘米× 5.72厘米宽× 6.35厘米深。坚持一个平面压克力板底部的形式。真空润滑脂密封所有的内部边缘。 </li><li>所需的几何实体模型（即身体，韧带，或浅固有层）的外侧端上放置一个真空润滑脂少量。按入模具形腔的底部，真空油脂的一面朝下的模式，使真空润滑脂持有到位。宽松外套模具形式和脱模剂的实体模型。使用油漆刷，确保脱模剂延伸到所有角落模具形腔。 </li><li>混合10 A部分和一个平滑SIL 950铂金硅橡胶在一个容器，它具有足够的扩展空间（按重量计算的部分）的B部分。删除与未固化的硅橡胶气泡的地方模具形式在真空室和减少压力（例如，低于大气压力的汞约26英寸），约三分钟（或必要的或多或少）。脱气硅，从室中取出，倒入模具形腔。将模具与固化的有机硅再次进入真空室和德加。从真空室和水平的表面上删除。允许治愈24小时，取出模具从模具形式。 </li><li>重复步骤1.1至1.4创建浅固有层，韧带，和身体层的每一个模具。 </li><li>削减韧带层模具在前后方向的直剃须刀，让纤维插入内侧表面的中心。 </li></ol> 2。铸造Øf每一层<ol><li> 体层 ：一层薄薄的涂上油漆刷脱模剂车身模具腔。混合B部分和一个环节的Ecoflex 00-30超柔白金硅（按重量计算）。加入其中的一部分硅胶稀释剂（重量比），以减少最终固化材料的刚度。混合在一起，在一分钟的真空室，除去夹带的空气30秒的地方。从真空中删除的混合物，倒入车身模具腔，但不填充整个模腔的顶部。烘箱中放置30分钟，在250 ° F。删除从烤箱和冷却。 </li><li> 基材 ：甲龙皮肤，并添加一个部分硅胶稀释剂（重量比）混合B部分和其中的一部分。大力搅拌30秒，1分钟的真空地点，并进入人体模腔倒，额满为止。烘箱中放置30分钟，在250 ° F。从烤箱和冷却模具。从模具中取出模型，让冷却到室温，并移除表面上的任何脱模剂身体用纸巾层。 </li><li> 韧带层 ：应用油漆刷上韧带模具型腔表面薄薄的一层脱模剂。将通过削减从直剃刀推入一个30厘米的线程，在模具。彻底混合B部分的Ecoflex 00-30四个部分有机硅稀释剂（重量比）的A部分。在真空室中，以去除气泡，倒入韧带模腔的混合物。 </li><li> 韧带层（续）：按到韧带模腔体支持模型（2.1.1和2.1.2步骤）。开始插入一侧，轻轻地移动到其他使模型推多余的固化的有机硅和气泡的模腔。如果气泡存在，删除从模腔，填充固化的硅模型，并重复压入模具模型。 30分钟的烤箱放置在模具，取出，冷却至室温。从模具中取出模型。删除多余的脱​​模剂，用纸巾。 </li><li> 浅固有层层 ：应用superifical固有层（SLP）模腔与油漆刷表面薄薄的一层脱模剂。混合一个B部分，一部分按重量更薄的Ecoflex 00-30，和8个部分硅一。真空完成以前，倒到SLP的模腔。使用相同的过程在步骤2.1.4中所述插入到浅固有层模腔韧带体后备模型。放入烤箱250℉，一小时的治疗。从烤箱中取出，并允许降温。慢慢取出模型格外小心，使浅固有层完好无损。 </li><li> 上皮细胞层 ：将声乐倍模型支持下的一个平坦的表面。取出一条直线剃须刀的支持材料。它们附加到对象模型的高度大于在空气中挂起线程。混合使用其中的一部分硅胶稀释剂，混合，真空B部分，其中一部分皮肤的龙，然后倒在模型上，并允许治愈Ë小时。重复该过程，创建一个层较厚。删除多余的材料用直剃刀。 </li><li> 可选 ：如果每一层都需要以不同的颜色（可见不同层检查），添加染料在混合过程中的Ecoflex或龙皮肤的B部分。 </li><li> 可选 ：如果将收集材料属性数据，同时创建制造的每一个模型层的拉伸强度和流变标本。额外的未固化材料注入模具脱模剂处理所需的材料属性试样的形状和大小。 </li><li> 可选 ：如果需要测量层的厚度，切一个截面直剃须刀模型，并用显微镜检查。 </li></ol> 3。最后测试模型的制备<ol><li>第一次申请的背面（横向）和侧面（一层薄薄的硅酮胶，丙烯酸安装板安装到每完成声带模型一个nterior后）模型表面。插入安装板的凹进切割模型。亚克力板的顶部对齐模型内侧表面。擦去多余的胶水。允许胶水固化一小时。 </li><li>滑石粉应用于模型表面，降低表面粘性。 </li><li>使用内侧表面跟踪的细点Sharpie笔到模型上的标记点。如果标记是应用滑石粉后出现最好的结果。 </li><li>通过对着现有的模型将被配对到的模型螺纹安装板孔长的螺栓。在螺栓莱线程。闭孔泡沫放置在螺栓关闭任何空气间隙。 </li><li>另一个同样已安装到使用步骤3.1和3.2丙烯酸持有人的声带模型配对准备模式。拧紧螺丝压缩泡沫和带来内侧表面直到达到预振动差距的愿望。确保线程都被放置OVER的螺栓和在前后方向的压克力板向外延伸。 </li><li>山声带对送风管上。 </li><li>铁前线程在一起，形成一个循环。重复后的线程。坑所需的重量同时对循环。 </li><li>模型现在准备测试和数据收集。 </li></ol> 4。代表性的成果使用这种制造工艺创建一个模型的振动响应数据如下;这些结果是典型的。张力约31克纤维，发病压力400宾夕法尼亚州在10％以上发病压力（440帕）的声门下压力，振动模式与声门的210毫升/秒流量在115赫兹人（ 见表3）报告的值，这些值吻合良好。使用高速videokymography分析模型运动之间优越的一个相区别的证据表明ND伪劣利润率，即上缘下缘隐蔽在开放阶段的振动周期（ 图3）。从内侧和下声带模型表面的点的立体图像中提取的轨迹表明，模型展示交替的汇聚发散的配置文件，是人类的发声，粘膜的波浪状运动，和较低的典型劣势卓越的运动比以前的型号（ 图4） 。 <img alt="表1。" src="/files/ftp_upload/3498/3498table1.jpg" /> 表1。模型的几何参数值。 <img alt="表2。" src="/files/ftp_upload/3498/3498table2.jpg" /> 表2。重量以及由此产生的杨氏模量声带模型的个别路段的混合比例。 EF和DS候硅制成的Ecoflex和龙皮分别为14。 <img alt="表3。" src="/files/ftp_upload/3498/3498table3.jpg" /> 表3。人力和合成声带振动响应之间的比较。 <img alt="图1。" src="/files/ftp_upload/3498/3498fig1.jpg" /> 图1。人造声带模型制作过程。 （左图）的CAD派生的实体模型，用于创建模具（中心面板），每一层。每一层再投，开始同体层和上皮细胞层（右面板中，每个“去皮”回能见度层）结束。模型制作完毕后，安装压克力板进行测试。 <img alt="图2。" src="/files/ftp_upload/3498/3498fig2.jpg" /> 图2。人造声带模型的横截面。不同的身体，浅固有层，LIG柔夷花序，和上皮细胞层。参数定义声带模型的几何形状。这个数字缩放几何定义明确表示。 表1中给出的参数值的应用将导致比略有不同的形状，这里显示的是什么。 <img alt="图3。" src="/files/ftp_upload/3498/3498fig3.jpg" /> 图3。模型振动的高速记波。彩色虚线所示的位置上，下边距的预算。伪劣和优越的利润率之间的相位差异是显而易见的。 <img alt="图4。" src="/files/ftp_upload/3498/3498fig4.jpg" /> 图4。合成声带模型在hemilarynx安排的时间在两个不同的实例捕获振动时，内侧的表面轮廓。墨水标记被放置在内侧表面（以sh自己在右边的图像），成像采用两个同步高速摄影机，振动周期跟踪。左边的图显示在开幕式阶段收敛声门和右边的图显示了一个在闭幕阶段的发散声门。

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作者什么都没有透露。

这种方法制造合成声带模型产量模型，表现出类似人类的声带的振动行为。多层次的概念，结果在减少发病的压力和改进模型运动方面的显著优势超过以前的一，二层模型设计3,6,8,15，（会聚发散振荡，粘膜波浪状运动期间的形象，并减少劣质优越的位移）。这里介绍的方法是表明一个有点理想化的模型，在几何方面，但它可以适用于不同的几何形状的模型。例如，对人体成像的几何数据...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;目录编号</strong</td&gt;<td<strong&gt;评论</strong</td&gt;</tr&gt;<tr&gt;<td&gt;高真空润滑脂</td&gt;<td&gt;道康宁</td&gt;<td&gt; 01018817</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; POL - EASE 2300</td&gt;<td&gt; Polytek</td&gt;<td&gt; POL - Ease2300 - 1</td&gt;<td&gt;脱模剂</td&gt;</tr&gt;<tr&gt;<td&gt;光滑银950</td&gt;<td&gt;平滑</td&gt;<td&gt;光滑银950</td&gt;<td&gt;模具制造材料</td&gt;</tr&gt;<tr&gt;<td&gt;真空泵</td&gt;<td&gt;爱德华兹</td&gt;<td&gt; E2M2</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;真空室</td&gt;<td&gt; Kartell的</td&gt;<td&gt; 230</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;压力表</td&gt;<td&gt;沼泽Bellofram</td&gt;<td&gt; 11308252A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;直线型刀片</td&gt;<td&gt;赫斯基</td&gt;<td&gt; 008 - 045 - HKY</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;的Ecoflex 00-30</td&gt;<td&gt;平滑</td&gt;<td&gt;的Ecoflex 00-30</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;硅胶稀释剂</td&gt;<td&gt;平滑</td&gt;<td&gt;硅胶稀释剂</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;龙皮肤</td&gt;<td&gt;平滑</td&gt;<td&gt;龙皮肤10快速</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;发新话题</td&gt;<td&gt;欧米茄</td&gt;<td&gt; OmegaCrys</td&gt;<td&gt;仅使用明确的纤维</td&gt;</tr&gt;<tr&gt;<td&gt;硅胶染料</td&gt;<td&gt;平滑</td&gt;<td&gt; SILC猪黑</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;硅酮胶</td&gt;<td&gt;平滑</td&gt;<td&gt;银，Poxy</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;滑石粉</td&gt;<td&gt;西方家庭</td&gt;<td</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

synthetic, multi-layer, self-oscillating vocal fold model fabrication

人类语音的声音是通过流引起的声带振动产生。声带由几层组织，各有不同的材料特性 1 。正常的语音生产依赖于健康的组织和声带，空气动力学，结构动力学和声学物理现象之间的复杂耦合的结果发生。语音障碍影响到750万，每年在美国就有2，往往会造成显著的财政，社会，和其他质量的生活困难。了解语音生产的物理学有可能显著受益语音服务，包括临床的预防，诊断，和治疗语音障碍。 现有的方法， 包括在研究语音生产的体内实验使用人类和动物的科目 ，在体外实验使用切除larynges和合成模型和计算模型ING。由于危险和困难的仪器访问，在体内实验中是极为有限的范围。切除喉实验解剖和某些生理现实主义的利益，但涉及几何和材料的属性变量的参数的研究是有限的。此外，他们通常只能是相对较短的时间周期（通常是为了分钟）振捣的。 克服部分切除喉实验的局限性，合成声带模式正在成为一个研究语音生产的辅助工具。可以制备合成模型与几何形状和材料性质的系统性变化，从而为人类健康和不健康的发声空气动力学，结构动力学，和声学研究。例如，他们已被用于研究左，右声带不对称3,4，临床仪器开发5，喉空气动力学6-9，VOC人接触压力10倍，并声门下声学11（更全面的清单中可以找到Kniesburges 等 12 ） 然而，现有合成声带模型，被同质（1层模型），或已采用两个不同刚度的材料（两层模型）。这种方法不允许起着核心作用声带流激振动响应的人类 1声带实际的多层结构的代表性。因此，一个和两个层合成声带模型已经表现出的缺点，如比什么是典型的人类发声（发病压力是最小的肺压力，要求启动振动），人为地大量劣质的高发病压力3,6,8卓越的运动，缺乏一个“粘膜波”（一个垂直行波的特点是健康人的声带振动）。 <pCLASS =“jove_content”&gt;在本文中，制造与多种不同的材料特性层模式的描述。层模型模拟人类的声带，包括上皮，浅固有层（SLP），中级和深固有层的多​​层结构（即，韧带，前后刚度包括光纤）和肌肉（即体）第1层。前结果，表明该模型展品改善振动特性和两层的合成模型，包括发病的压力更接近人类发病压力，减少劣质卓越的运动，和粘膜波的证据。

Watch this Scientific Journal Video about Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication at JoVE.com

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

用于制造合成声带模型的方法描述。该机型是等身和模仿人类声带的多层结构。结果表明，模型自振荡压力与肺压力和展示类似人类声带的流激振动响应。

合成，多层，自激振荡的声带模型制作

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

synthetic-multi-layer-self-oscillating-vocal-fold-model-fabrication

Research

JoVE Journal

Bioengineering

13.9K 次观看.  Brigham Young University. 用于制造合成声带模型的方法描述。该机型是等身和模仿人类声带的多层结构。结果表明，模型自振荡压力与肺压力和展示类似人类声带的流激振动响应。

视频: Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

科研

生物工程

Synthetic Spider Silk Production on a Laboratory Scale

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

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

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

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 ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

用于合成生物学和天然产物应用的高产 链霉菌 转录翻译工具包

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

用于癌症诊断的 CRISPR-Cas 合成尿液生物标志物测试

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

作者聚焦：用于疾病检测和成像的工程分子工具

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.