Name: single-cell microfluidic analysis of bacillus subtilis
Uploaded: 2018-01-26T14:30:00
Description: Watch this Scientific Journal Video about Single-cell Microfluidic Analysis of Bacillus subtilis at JoVE.com

该项目由国家卫生研究院资助, GM018568。该协议的一部分是在纳米系统中心 (CNS), 这是国家纳米技术协调基础设施网络 (NNCI) 的成员, 得到国家科学基金会的支持下, NSF 奖1541959。CNS 是哈佛大学的一部分。许多感谢是由于托马斯诺曼和内森勋爵的工作, 在构思和制作的主模板使用的设备, 在这里显示和建设的原始版本的设备。我们还感谢约翰 Paulsson 的宝贵合作意见, 感谢他的实验室成员的意见和不断改进的细菌微流控装置。

1. 硅橡胶器件铸件
<ol><li>在无尘环境中, 将聚氧烷聚合物和固化剂以10:1 的比例混合, 在真空下至少10分钟。</li>
	<li>在聚苯乙烯培养板上的一块完整的铝箔上放置一个硅母 (或其环氧或聚氨酯复制品)。倒入脱的硅橡胶混合物在大师到深度大约5毫米. 加气培养皿至少10分钟. 使用柔和的气流来打破表面气泡。 注意: 所使用的双层设备的尺寸在随附的 CAD 文件5中提供 (请参阅?...

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+microfluidic+tracking+of+coccoid+cyanobacterial+cells+reveals+robust+control+of+division+timing.">Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing.</a> BMC Biol. 15 (1), 11 (2017).</li><li>Campos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+constant+size+extension+drives+bacterial+cell+size+homeostasis.">A constant size extension drives bacterial cell size homeostasis.</a> Cell. 159 (6), 1433-1446 (2014).</li><li>Taheri-Araghi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-size+control+and+homeostasis+in+bacteria.">Cell-size control and homeostasis in bacteria.</a> Curr Biol. 25 (3), 385-391 (2015).</li><li>Siryaporn, A., Kim, M. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+prokaryotic+social+behaviors+with+bacterial+"lobster+traps".">Probing prokaryotic social behaviors with bacterial "lobster traps".</a> MBio. 1 (4), (2010).</li><li>Long, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+bacterial+adaptation+dynamics+at+the+single-cell+level+using+a+microfluidic+chemostat+and+time-lapse+fluorescence+microscopy.">Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy.</a> Analyst. 139 (20), 5254-5262 (2014).</li><li>Okumus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+slowing-down+of+cytoplasmic+diffusion+allows+in+vivo+counting+of+proteins+in+individual+cells.">Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.</a> Nat Commun. 7, 11641 (2016).</li><li>Teng, S. W., Mukherji, S., Moffitt, J. R., de Buyl, S., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+circadian+oscillations+in+growing+cyanobacteria+require+transcriptional+feedback.">Robust circadian oscillations in growing cyanobacteria require transcriptional feedback.</a> Science. 340 (6133), 737-740 (2013).</li><li>Baker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable,+Pneumatically+Actuated+Microfluidic+Device+with+an+Integrated+Nanochannel+Array+To+Track+Development+of+Individual+Bacteria.">Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria.</a> Anal Chem. 88 (17), 8476-8483 (2016).</li><li>Russell, J. R., Cabeen, M. T., Wiggins, P. 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这个微协议是灵活的, 因为许多步骤可以修改, 以优化其使用特定的物种或应变或为特定目的。实际上, 在本协议中, 我们对原始的 "母机器" 概念4进行了修改, 以优化其与B. 枯草-枯草杆菌的使用。通常, 细胞被限制的沟槽构成一个单层特征, 而在这个协议中我们使用两层细胞沟, 在细胞周围有一个浅通道。两层设计被引入, 作为一种方法, 最大限度地从B. 枯草细胞的养?...

地球上最引人注目的生命特征之一就是它的韧性和多样性。分子生物学的一个中心目标是理解细胞利用基因和蛋白质在各种各样的环境条件下最大化生长和适应的逻辑。为了实现这一目标, 科学家必须能够自信地观察单个细胞在特定条件下如何生长、分裂和表达基因, 并注意到细胞对其环境的后续变化的反应。然而, 传统的微生物学分析方法有技术上的局限性, 影响到可以解决的问题的类型。例如, 基于批量区域性的分析在过去的几年中非常有用, 但它们只提供了能够掩盖有意义的细胞间变异或细胞在总人口中的较小的子群的行为的人口级数据。基于光显微镜的活细菌单细胞分析揭示单细胞行为, 但在技术上也是有限的。细菌通常固定在含有生长培养基的琼脂糖垫上, 但细胞生长和分裂会使显微镜下的观察和消耗的养分在短短的几个细胞周期后耗尽, 极大地限制观测时间1, 2。此外, 由于细胞生长而导致的营养物质的局部耗竭和代谢副产物的积累, 不断地改变着当地细胞生长的环境, 难以测量或预测。使用琼脂糖垫的这种环境变化对稳态行为的研究或对生长条件下的特定变化的细胞反应都是一个挑战3。
微流体技术, 其中一个液体介质是不断流经 microfabricated 设备, 提供了一个经典的实验限制的解决方案。微流控装置可以保持单个细胞在活细胞显微镜中的位置, 而生长介质的流动不断地为细胞提供新鲜的养分, 洗去代谢副产物和多余的细胞, 从而产生高度均匀的生长环境.在恒定生长条件下, 细胞行为可以从环境因素的影响中分离出来, 从而使细胞内部逻辑得以不受阻碍的观察。当流体流动防止微流控装置变得拥挤与细胞时, 单细胞血统观察为数以万计或数百个世代成为可能4,5。如此长的观察时间允许检测到其他不可察觉的长期或稀有的细胞行为。最后, 通过该装置流动的培养基的组成可以随意改变, 使细胞在对压力的发生或对某一化合物的引入或去除作出反应时能够被观察到。
微已经享受了许多重要的应用。例如, 它已被用于组织, 器官, 或身体上的芯片设备, 其中多个人类细胞类型的共同培养, 以模拟在体内条件6;微环境中线虫运动的研究7;检查细菌生物膜之间的相互作用 (例如, 8);并用于封装和操作微小体积的单元或化学物质 (例如, 9)。微流控器件在微生物学领域也越来越受欢迎 (对于优秀的评论, 请参见10和11), 特别是由于它们的物理和流特性与自然微生物龛位的匹配很好,12。例如, 微最近被微生物学用于精确测量细胞生长和分裂13,14,15, 分析病原体移动16,监视仲裁传感17和生理过渡18, 以及蛋白质计数19, 在许多其他示例中。这里提出的方法是专门为分析单一细菌细胞的血统, 而不是组合的菌株或物种。此处演示的微流控装置利用了 "母机器" 设计的一个变体4, 其中的单元格在一个封闭端和一个开放端的微流控槽内的单文件中生长;细胞生长和分裂将子代细胞向上和流出的开放端推向流体流动。我们的分析通常只着眼于 "母亲" 的细胞, 被限制在沟的封闭端。我们认为这种方法是对以往基于光镜的单细胞分析技术的进步, 例如琼脂糖垫上的细胞固定。在这里, 当枯草芽孢杆菌被用作模型时, 该方法也适用于其他细菌种类 (大肠杆菌是另一种常见的模型; 某些具有不同细胞大小或形态的物种可能需要制造新的设备不同的尺寸)。使用荧光记者来标记细胞和可视化基因表达的变化需要使用基因驯服的物种;然而, 即使没有荧光标记, 对细胞生长和形态学的分析也是可能的。
本议定书不包括使用光刻技术制作硅母版的过程, 它在别处被广泛描述为5;硕士也可以很容易地外包的微细加工设施。它包括从增强硅母料中成型一个多晶硅装置;将装置与盖板玻璃接合;组装微流控入口和出水管道, 包括夹紧阀, 允许中型开关;钝化该装置, 制备细菌细胞, 并将该装置装入细菌细胞;将管道连接到设备上并均衡电池;并将该装置装入荧光显微镜进行成像。由于许多不同的图像采集和处理软件工具可以用来可视化和分析不同的数据的兴趣4,5, 示例图像显示, 但图像捕获方法不包括在此协议中。

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">(240)4019862</td>
			<td style="width:167px;">This is referred to as PDMS in the protocol. There are 2 components that are mixed at a 10:1 ratio</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">0.75-mm biopsy punch</td>
			<td style="width:71px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">22 x 40 mm No. 1.5 cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48393 172</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Plasma Etch PE-50</td>
			<td style="width:71px;">Plasma Etch Inc.</td>
			<td style="width:119px;">PE-50</td>
			<td>Instrument used to bond PDMS to glass using oxygen plasma treatment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Tygon flexible tubing ID 0.02&#34;, OD 0.06&#34;</td>
			<td style="width:71px;">Saint-Gobain PPL Corp.</td>
			<td>AAD04103</td>
			<td>For the main part of the tubing, e.g. attached to the needles</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:223px;">Silicone Tubing, 0.04&#34; ID, 0.085&#34; OD</td>
			<td style="width:71px;">HelixMark Standard Silicone Tubing</td>
			<td style="width:119px;">60-795-05</td>
			<td>For pinch valves and Y-junction connection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Bovine Serum Albumin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906-100G</td>
			<td>Used as passivation agent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">ART Gel Pipet Tips (P200)</td>
			<td style="width:71px;">Molecular BioProducts</td>
			<td align="right" style="width:119px;">2155</td>
			<td>For loading the device with medium and cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Acrodisc 32-mm syringe filter with 5-&#956;m Supor membrane</td>
			<td style="width:71px;">Pall Life Sciences</td>
			<td align="right" style="width:119px;">4560</td>
			<td>For filtering cultures before device loading</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">21-ga blunt needles, 1&#34;</td>
			<td style="width:71px;">McMaster-Carr</td>
			<td style="width:119px;">75165A681</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Y connector with 200-series barbs, 1/16&#34; ID tubing, polypropylene</td>
			<td style="width:71px;">Nordson Medical</td>
			<td style="width:119px;">Y210-6005</td>
			<td>The company is AKA Value Plastics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eclipse Ti-E inverted microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>This unit has been discontinued by the manufacturer and is replaced by the Ti 2 E.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">LB Lennox</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Microfuge 18</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td align="right">367160</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Bacillus subtilis strain NCIB3610</td>
			<td style="width:71px;">Bacillus Genetic Stock Center</td>
			<td style="width:119px;">3A1</td>
			<td>wild-type parental strain modified for use in the experiments shown in this protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Gravity convection oven</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">414005-106</td>
			<td>for curing PDMS and baking assembled devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch-Weld Epoxy Kit</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">2216 B/A</td>
			<td>may be used to bond a silicon wafer to an aluminum backing plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Scotch Magic tape, 3/4&#34; width</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;"></td>
			<td>to clean dust from PDMS devices and to place over features to increase visibility (denoted &#34;office tape&#34; or &#34;adhesive tape&#34; in protocol)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Stereomicroscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">SMZ800N</td>
			<td>listed here as an example; nearly any stereomicroscope will do</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isopropyl alcohol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">W292907</td>
			<td>To clean cover glass</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Syring pump, 6 channel</td>
			<td style="width:71px;">New Era Pump Systems Inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Kimwipes</td>
			<td style="width:71px;">Kimberly-Clark</td>
			<td align="right" style="width:119px;">34120</td>
			<td>a brand of dust-free wipes</td>
		</tr>
	</tbody>
</table>

single-cell microfluidic analysis of bacillus subtilis

微流控技术克服了传统的微生物学分析方法的许多局限性。与大块培养方法不同, 它提供单细胞分辨率和长达数百代的长观测时间;不同于琼脂糖垫基显微镜, 它有统一的生长条件, 可以严格控制。由于生长培养基的连续流动使微流控装置中的细胞与细胞生长和新陈代谢引起的局部化学环境的不可预知的变化隔绝, 基因表达和细胞生长的真实变化反应具体的刺激可以更自信地观察。枯草芽孢杆菌在这里用作细菌模型, 以演示 "母机器" 型细胞分析方法。我们展示了如何构造和铅化一个微流体设备, 加载它与细胞, 启动显微成像, 并暴露细胞的刺激, 从一个生长媒介切换到另一个。一个压力响应的记者被用来作为一个例子来揭示这种方法可以获得的数据类型。我们还简要地讨论了这种方法在其他类型的实验, 如细菌产分析的进一步应用。

在将微流体管道连接到设备之前, 通过相衬显微镜评估的成功的初始细胞负荷, 将被认为具有包含一个或多个细菌细胞的所有或几乎所有的微流体侧通道 (图 3A)。最佳加载将显示每个通道中的多个单元格, 但是通道将在平衡期间 (图 3B) 中由于单元格的增长而填充单元格。有较差负荷的车道, 其中相对较少的 (< 50%) 侧通道装有细胞,...

Watch this Scientific Journal Video about Single-cell Microfluidic Analysis of Bacillus subtilis at JoVE.com

Single-cell Microfluidic Analysis of Bacillus subtilis

单细胞微流控芯片分析枯草芽孢杆菌

本文以枯草芽孢杆菌为例, 对单个细菌细胞谱系的微流体分析方法进行了研究。该方法克服了传统分析方法在微生物学中的不足, 允许在严格控制和均匀生长条件下观察几百个细胞世代。

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Research

JoVE Journal

Biology

Single-cell Microfluidic Analysis of Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

科研

生物学

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Single-cell Microinjection for Cell Communication Analysis

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

单探头的应用：根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

大肠杆菌抗生素持久性的群体和单细胞分析

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...