Name: assembly and tracking of microbial community development within a microwell array platform
Uploaded: 2017-06-06T15:30:00
Description: Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

在美国能源部基础能源科学办公室的纳米相材料科学用户设备部中心制造和表征微孔阵列。通过橡树岭国家实验室主任的研发基金提供了对这项工作的财政支持。作者还要感谢J.Mougous实验室（华盛顿大学西雅图华盛顿州）提供这些研究中使用的铜绿假单胞菌菌株。

硅微孔阵列制造<ol><li> 聚对二甲苯涂层 <ol><li>根据制造商的规格和说明书（设置：蒸发器设定点= 160℃;炉设定点= 650℃），使用市售的聚对二甲苯涂层系统在硅晶片上沉积1-1.5μm的聚对二甲苯N. 注意：装入室内的聚对二甲苯约6克产生1-1.5微米厚的涂层。 </li></ol></li><li> 光刻 <ol><li>以3,000rpm转速涂覆具有粘合促进剂，20％六甲基二硅氮烷（HMDS）和80％丙二醇单甲醚乙酸?...

<ol>
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本文介绍了微孔阵列设备和实验方案，旨在实现基于细胞群发展的高通量和高含量的基于活细胞成像的分析。虽然这里的示范重点是研究接触介导的VI型分泌对社区发育的影响，但阵列被设计为灵活的，适应于广泛的微生物群落和微生物 - 微生物相互作用的研究。这里的工作仅在于使用组成性表达荧光标记物的细菌，以便容易地跟踪成员丰度和位置。然而，通过改变化学环境，通过生长或扰动后?...

微生物群落由细胞死亡，分裂，蛋白质浓度，细胞器数目和突变1相关的环境结构和随机过程两个决定因素构成。在自然环境中，几乎不可能分析这些影响对社区组成和活动的个人影响。被自然结构遮蔽，埋在化学和生物环境中，识别社区成员并进一步解决其在自然环境中的时空分布是极具挑战性的。尽管如此，最近的努力已经强调了空间组织对社区功能的重要性，并指出在正在进行的研究2,3,4中需要考虑成员丰富和组织。 它很清楚，当地的化学环境（ 即营养物质和次生代谢物的可获得性），物理结构（ 如土壤结构，植物根系，海洋颗粒或肠道微绒毛），氧气的存在或不存在以及引入致病物种都影响微生物群落5,6,7,8,9,10,11的组成，结构和功能。然而，忽视捕捉这些因素的传统文化技术依然存在。社区组成（ 例如，共同依赖物种的存在），物理附着，信号分子浓度和直接的细胞 - 细胞接触都是形成微生物群落的重要因素，并且可能在c传统文化条件。这些性质难以在大量液体培养物或琼脂平板上复制。然而，允许复制自然环境的关键物理和化学特征的微流体，微图形化和纳米制造技术的可用性使得许多研究人员能够建立细菌群落来研究它们的相互作用12,13,14 ，并开发合成环境模拟自然条件4，15，16，17，18，19，20。 该协议描述了一种制造微孔阵列器件的方法，并提供了可用于功能化th的详细实验程序e井在阵列中生长细菌，作为单一物种殖民地和多成员社区。这项工作还演示了如何使用细菌修饰以产生荧光报告蛋白可以随时间监测孔内的细菌生长。以前提出了类似的阵列，并且显示可以在微孔中跟踪绿脓假单胞菌（ 铜绿假单胞菌）单种群的生长。通过调节孔径和接种密度，数千个生长实验的起始条件可以并行变化，以确定初始接种条件如何影响细菌生长的能力21 。目前的工作使用微型阵列的稍微修改版本，其基于以前的工作，通过实现多个阵列的同时比较和使用更强大的实验协议。此工作中使用的数组包含多个子阵列或阵列包含不同尺寸的直径为15-100μm的孔，其以三个不同的间距（ 即孔径的2x，3x和4x）排列。将阵列蚀刻到硅中，并且通过用已经用中等浸渍的琼脂糖凝胶涂覆的盖玻片密封阵列来实现在硅阵列中接种的细菌的生长。用于研究VI型分泌系统的铜绿假单胞菌突变体用于该示范。 这里提出的结果构建在分析微孔阵列中的多元群落的最终目标，使研究人员能够在控制和探测化学环境的同时监测原位细菌的丰度和组织。这应该最终提供洞察管理社区发展和继承的“规则”。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parylene N</td>
			<td>Specialty Coating Systems</td>
			<td>CAS NO.:1633-22-3</td>
		</tr>
		<tr>
			<td>Parylene coater</td>
			<td>Specialty Coating Systems</td>
			<td>Labcoter 2 Parylene Deposition Unit PDS2010</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>WRS Materials</td>
			<td>100mm diameter, 500-550&#956;m thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;,</td>
		</tr>
		<tr>
			<td>adhesion promoter</td>
			<td>Shin-Etsu Microsci</td>
			<td>MicroPrime P20 adhesion promoter</td>
		</tr>
		<tr>
			<td>postive tone photoresist</td>
			<td>Rohm and Haas Electronics Materials LLC (Owned by Dow)</td>
			<td>Microposit S1818 Positive Photoresist (code 10018357)</td>
		</tr>
		<tr>
			<td>Quintel Contact Aligner</td>
			<td>Neutronix Quintel Corp</td>
			<td>NXQ 7500 Mask Aligner</td>
		</tr>
		<tr>
			<td>Reactive Ion Etching Tool</td>
			<td>Oxford Instruments</td>
			<td>Plasmalab System 100 Reactive Ion Etcher</td>
		</tr>
		<tr>
			<td>R2A Broth</td>
			<td>TEKnova</td>
			<td>R0005</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>Enspire, 2300-0000</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
		</tr>
		<tr>
			<td>Automated Stage</td>
			<td>Prior</td>
			<td>ProScan III</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Nikon</td>
			<td>DS-QiMc</td>
		</tr>
		<tr>
			<td>Stage-top environmental control chamber</td>
			<td>In Vivo Scientific</td>
			<td>STEV ECU-HOC</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
		</tr>
		<tr>
			<td>25 x 75 mm No. 1.5 coverslip</td>
			<td>Nexterion</td>
			<td>High performance #1.5H coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence Reference Slides</td>
			<td>Ted Pella</td>
			<td>2273</td>
		</tr>
		<tr>
			<td>Physical Stylus Profilometer</td>
			<td>KLA Tencor</td>
			<td>P-6</td>
		</tr>
		<tr>
			<td>lab wipes</td>
			<td>Kimberly Clark</td>
			<td>Kimipe KIMTECH SCIENCE Brand, 34155</td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Nikon</td>
			<td>NIS Elements</td>
		</tr>
		<tr>
			<td>Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>filter cubes</td>
			<td>Nikon</td>
			<td>Nikon FITC (96311), Nikon Texas Red(96313)</td>
		</tr>
	</tbody>
</table>

assembly and tracking of microbial community development within a microwell array platform

微生物群落的发展取决于复杂的确定性和随机因素的组合，可以显着改变社区成员的空间分布和活动。我们开发了一个微孔阵列平台，可用于并行快速组装和跟踪数千个细菌群落。该协议强调了该平台的实用性，并描述了其用于光学监控平台内阵列集合内简单的两人社区的发展。该示范使用铜绿假单胞菌的两个突变体，一系列突变体的一部分用于研究VI型分泌致病性。 mCherry或GFP基因的染色体插入片促进具有不同发射波长的荧光蛋白的组成型表达，可用于监测每个微孔内的群落成员丰度和位置。该协议描述了详细的方法d用于将细菌混合物组装到阵列的孔中，并使用延时荧光成像和定量图像分析来测量每个成员群体随时间的相对增长。微孔平台的种植和组装，阵列内微生物群落定量分析所需的成像过程以及可用于揭示微生物物种区域之间相互作用的方法。

这里提出的实验平台是为细菌群落的高通量和高含量研究而设计的。该设计使得能够同时分析成千上万个种植在各种尺寸的井中的社区。通过这种微孔阵列设计，可以确定最终社区组成对初始播种密度，孔尺寸和化学环境的依赖性。这项工作展示了微型阵列中两个成员社区的发展，并提出了分析社区组成和组织的方法。 这里?...

Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微生物群落的发展取决于环境结构，成员丰度，特征和相互作用等因素的结合。该协议描述了一种合成的微制造环境，用于同时跟踪包含在femtoliter井中的数千个社区，其中可以近似关键因素，如利基尺寸和约束。

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

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SCIENTISTS IN ACTION

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

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生物工程

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Development of an In Vitro Ocular Platform to Test Contact Lenses

的发展<em&gt;体外</em&gt;眼平台，试验用隐形眼镜

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Micropatterning and Assembly of 3D Microvessels

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

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 ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

大鼠运动皮质微电极植入所致功能缺损的啮齿动物行为检测

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...