Name: single-cell microfluidic analysis of bacillus subtilis
Uploaded: 2018-01-26T14:30:00.000+00:00
Duration: 10 min 37 s
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 (请参阅补充文件 1)。在脱气过程中, 塑料复制主可以部分浮动;如果发生这种情况, 用一个干净的塑料涂抹器向下推复制副本。</li>
	<li>在60° c 的烤箱中固化至少1小时, 冷却至室温。从培养皿中取出含有该母材的铝箔和硫化的硅橡胶。小心剥去主人背上的铝箔, 然后小心地剥去主人的皮。 注: 另外, 在室温下, 可以在一夜之间治愈。硅晶片的相对脆弱性可以克服使用环氧树脂胶粘剂永久债券的硅片, 以1/16 英寸厚的铝盘大小相同的硅片。</li>
	<li>由于晶圆通常包括几种微流体设备, 尺寸略有不同 (请参阅补充文件 1), 使用干净、锋利的手术刀将单个设备切割成尺寸。 注意: 对于例程B. 枯草, 请使用具有1.0 µm 宽的单元格沟槽的设备, 在随附的 CAD 文件中用 C 或 F 标记。</li>
</ol>2. 设备冲压和粘合
<ol><li>在设备的图案一侧放置一条半透明的办公室磁带 (请参见材料表), 以增强图案化功能的可见性。入口和出口端口被确定为可见的射流通道两端的圆形区域 (图 1)。为了提高入口和出口引脚在设备上冲孔时的能见度, 请使用细尖标记标记入口和网点。 注意: 为了确保入口和出口引脚不拥挤, 每个其他通道都被打上, 从该设备的字母指示符下面的通道开始。</li>
	<li>翻转的硅橡胶设备, 使图案的一面是下来, 并通过该设备打孔通过0.75 毫米活检冲床的标记点;丢弃 punchings。 注: 由于活检穿孔所产生的孔通常在穿孔进入聚合物的地方略有爆发, 所以设备被打成图案的一侧。以倒置的方向冲孔设备可确保图案一侧的孔具有锋利的边框。</li>
	<li>使用显微镜, 确保穿孔孔与设备的入口和出口区域重叠。使用细尖的镊子, 以消除任何外来的碎片, 从穿孔孔。</li>
	<li>使用胶带去除设备两侧的灰尘, 放入干净的聚苯乙烯培养板。用100% 异丙醇润湿22毫米 x 40 mm 盖玻璃, 并用无尘擦拭擦;用无尘的空气或氮气流干。</li>
	<li>将穿孔的硅橡胶设备功能端与清洗过的盖玻璃一起放到氧气等离子清洗器中。<ol>
<li>等离子处理设备的设置如下: 真空, 70 mTorr;O2压力, 200 mTorr;期间, 十五年代;电源, 30 w 在等离子处理期结束后立即取出设备并将玻璃从等离子清洗器上盖上。</li>
		<li>倒置的硅橡胶, 并把它放在封面玻璃的中心, 使图案的一面接触玻璃;确保进料通道 (在入口和出口区域之间运行) 与盖玻璃的长轴对齐。按下非常轻轻, 以确保对玻璃的硅橡胶密封。</li>
	</ol>
</li>
	<li>在烤箱中烘烤至少1小时60° c 的组装装置。烘烤后, 通过在设备的一个角落轻轻推送, 以手动验证该产品是否与玻璃结合。 注: 一旦设备粘合, 应在24小时内使用, 因为聚合物将变得越来越疏水性随着时间的推移, 等离子体处理。</li>
</ol>3. 微流体管道的制备
<ol><li>聚合物管材的切割长度 (内径 (ID) 0.02 英寸, 外径 (外径) 0.06 英寸) 到适当的长度, 以达到从注射器泵到开关设备 (如果使用) 和设备时, 安装在显微镜上。切割尽可能多的长度, 因为有微流体通道。</li>
	<li>通过切割和附加2厘米长的挠性硅胶油管 (0.03 英寸 ID, 0.065 英寸外径) 到 "y" 和1厘米长的硅胶油管到底部的 "y" 的倒钩上, 为夹紧阀装配 y 接头 (如果使用)。</li>
	<li>切割10厘米 (或适当) 长度的聚合物管材;通过将它们插入到 "Y" 的底部倒钩上的1厘米硅管段, 将它们一端连接到开关接合处。连接他们在另一端的21G 针已经从他们的塑料注射器适配器和弯曲大约90°大约9毫米从针末端被去除。</li>
	<li>连接21G 钝针到油管段的一端, 从注射器到开关装置, 将针头插入油管。将各油管长度的另一端插入硅胶导管段, 在 Y 形路口的入口倒钩上。 注: 使用某种仪器来控制夹紧阀和接合处;这可以很容易地从一个微提示框 (图 2D)。</li>
	<li>切断聚合物导管的长度, 将废料从装置的出口处运到废物烧杯。连接他们在一端与弯曲的21G 针如上所述准备。把管子的另一端带到一个废弃的烧杯里。</li>
	<li>准备适当数量的培养基, 含0.1 毫克/毫升牛血清白蛋白 (BSA), 并填充所需的注射器大小和数量。将 BSA 加载的注射器连接到所准备的21G 针上, 并将注射器装入注射器泵中。 注: 对于典型的实验, 使用了5通道, 每台都有20毫升的注射器。一个实验, 其中的媒介是交换将需要2银行5注射器每。在这里, 包含第一个银行的泵被称为 phase-1 泵, 包含第二个银行的泵, 与后交换介质, 被称为 phase-2 泵。</li>
	<li>通过在高流速下运行注射器泵 (> 500 µL/分钟), 从入口管道中清除空气, 首先将注射器垂直定向, 然后敲击注射器, 使气泡顶部。一旦空气已从注射器中清除, 放置注射器泵水平, 并逐步轻拍或轻弹的聚合物线从注射器通过开关装置, 以消除和清除气泡。对第二个注射器 (如果切换介质) 重复此过程。</li>
	<li>清除气泡后, 设置 phase-2 注射器泵 (即, 用将在开关后使用的介质) 转换为1.5 µL/分钟, 然后暂停流。将小活页夹夹在与第二介质相对应的 Y 接合处的挠性油管段上。继续运行 phase-1 注射器泵在一个适度的流速 (例如, 10 µL/分钟) 至少30分钟, 以清除第二个介质从进口油管下游的 Y 交界处。</li>
</ol>4. 细胞培养制剂
<ol><li>在一夜之间, 在所需的培养基中生长出培养的兴趣, 到固定相。细菌的菌株应包含一个突变, 使细胞不动, 使细胞不游出的渠道的设备。 注: 在本协议中, 细菌菌株为B. 枯草3610, 包含巫婆A233V替换 (此突变导致鞭毛为直而不是螺旋状, 防止细胞运动), PrsbVmNeonGreen记者的细胞压力, 和本组织的 Phyperspank-mNeptune (红色) 记者突出细胞。在示例协议中使用了 LB 蓝诺克斯介质。典型的微实验菌株包含一个或多个荧光转录的记者和一个组成产生的细胞质荧光蛋白, 以促进细胞的自动检测。当使用了富培养基时, 未观察到生长缺陷, 但在微流体条件下, 由于分泌的铁被介质流冲走, 所以在最小介质中的枯草芽孢杆菌的生长可能受到抑制。在这种情况下, 可以通过添加柠檬酸钠和氯化铁来恢复培养基, 如 S750介质11所报告的那样。</li>
	<li>在实验的早晨, 将枯草芽孢杆菌细胞稀释1:50 成一个250毫升的、含有25毫升生长培养基的瓶子。在37° c 的震动培养中, 将细胞生长为 5-6 h, 光学密度大于1。 注: 稠密的细胞培养是可取的, 因为固定阶段的B. 枯草细胞更小, 在细胞链中发现较少, 便于设备加载。不同的细菌种类可能需要其他培养基或生长条件以获得最佳的负荷。</li>
	<li>使用装有5µm 孔径过滤器的注射器, 将大约15毫升的细胞培养物过滤到15毫升的锥形管中, 以移除B. 枯草杆菌细胞链 (对于大肠杆菌是不必要的, 而且可能不需要与其他物种)。 注: 应删除相对较少的细胞;滤液应该混浊。</li>
	<li>离心过滤培养10分钟在室温 4000 x g。倒入上清液, 重残留在管中的剩余上清液体中的细胞颗粒, 如有必要, 增加大约500µL 的新鲜培养基, 以促进移细胞悬浮。</li>
</ol>5. 设备加载
<ol><li>轻轻地将空的薄凝胶加载微提示 (请参见材料表) 放入微流体设备的出口孔中。</li>
	<li>使用 P200 微的薄凝胶加载技巧, 钝化该装置通过注入含有1毫克/毫升 BSA 的介质进入入口孔, 观察出口孔中的提示, 以监测微流控通道的填充 (图 2A)。在室温下孵育设备约5分钟。 注意: BSA 通常用作阻挡或钝化剂, 它将结合到聚脂聚合物的疏水性表面, 增加其亲水性, 减少其他蛋白质或细胞 (通过细胞表面分子) 的结合。</li>
	<li>使用薄的凝胶加载技巧, 加载每个通道与悬浮单元 (从4节), 使用插座通道中的提示, 通过设备监视单元的进度 (图 2B)。 注: 通过将 P200 微设置为其最大 200-µL 体积, 然后在吸进小体积 (大约一半的吸管尖端薄部分的体积) 之前, 将空气中的气垫吸进。悬浮细胞的体积不需要精确, 因为该设备的体积相对于微。我们知道没有上限的密度的悬浮细胞, 只要细胞悬浮可以 pipetted 到设备。细胞团块可以堵塞的设备, 应避免通过彻底移和/或涡混合。</li>
	<li>轻轻地从出水孔中取出凝胶加载的小贴士, 一次工作一次以防止损坏设备。</li>
	<li>离心机在一个适当的转子适配器在一个合适的离心在大约 6000 x g 10 分钟 (图 2C) 的设备。 注: 采用定制加工的铝转子适配器, 设计成适合离心转子 (图 2C);不同的离心机模型可以使用适当的转子适配器。该适配器的重要特点是, 它提供了横向力量的方向, 封闭两端的细胞战壕, 从而迫使细胞进入侧通道。</li>
	<li>验证显微镜下的成功加载 (图 3), 但不将设备粘贴到幻灯片持有者/阶段插入。 注: 在这个协议中, 一个 60X, 1.4 NA Ph3 油浸泡目标是用于在这一阶段的验证和后续成像。</li>
</ol>6. 设备装配、平衡和安装
<ol><li>小心地将已加载的设备安装到舞台插入上, 方法是: 将该系统的两侧的盖玻璃贴在舞台插入的底部 (即, 在附加设备之前反转舞台插入)。反转舞台插入设备组件, 使其在软表面上朝向并放置, 如无尘擦拭 (图 2D)。</li>
	<li>将 phase-1 注射器泵的流量调整到35µL/分钟, 每次使用一条车道, 插入进口针, 然后将出口针 (即, 运行到废物烧杯;图 2E)。</li>
	<li>用干净的无尘擦拭擦去多余的介质, 并目测检查设备是否有泄漏。检查排气管的外观过剩的细胞 (通常出现的混浊条纹) 和中型半月板, 这将慢慢地向废物烧杯。</li>
	<li>允许设备运行在35µL/分钟约 15-30 分钟, 直到所有连接的车道排入废物烧杯。</li>
	<li>设置 phase-1 注射器泵到1.5 µL/分钟, 并暂停流。将整个泵和装置设备带到显微镜 (这一过程是通过把它全部放在一个滚动的推车), 并重新启动 phase-1 介质流量在1.5 µL/分钟. 小心地安装在倒置荧光显微镜上的舞台插入装置上, 使用必要的胶带, 以路线的进口和出口油管。</li>
	<li>在设备上定位所需位置以进行成像, 并根据需要开始成像。请注意, 细胞通常需要几个小时 (大约10代) 达到稳态指数阶段的增长, 并开始的图像采集可能会延迟的期望, 使成像从平衡期之后。 注意: 在随后的图像分析中, 应通过跟踪细胞分裂时间和确保它们达到恒定的最小值来验证在指数阶段保持的细胞的稳态生长。</li>
</ol>7. 中型开关
<ol><li>在连续的一轮成像, 暂停流动的 phase-1 注射器泵。仔细松开的粘合剂剪辑从 phase-2 硅胶油管, 移动每一个硅胶管在 phase-1 分支的 Y 交界处。暂停流动的 phase-2 注射器泵 (这是设置为1.5 µL/分钟在步骤 3.8), 并继续成像。 注: 在1.5 µm/分钟与10厘米长的聚合物油管的 Y 结的下游, 第二阶段的介质到达设备在大约50分钟。该设备的时间可以通过使用含有荧光示踪粒子的介质进行经验测试。</li>
</ol>

<ol>
	<li>Moffitt, J. R., Lee, J. B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+an+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab Chip. 12 (8), 1487-1494 (2012).</li><li>Young, J. W., 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+single-cell+gene+expression+dynamics+in+bacteria+using+fluorescence+time-lapse+microscopy.">Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy.</a> Nat Protoc. 7 (1), 80-88 (2011).</li><li>Dusny, C., 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=Technical+bias+of+microcultivation+environments+on+single-cell+physiology.">Technical bias of microcultivation environments on single-cell physiology.</a> Lab Chip. 15 (8), 1822-1834 (2015).</li><li>Wang, P., 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=Robust+growth+of+Escherichia+coli.">Robust growth of Escherichia coli.</a> Curr Biol. 20 (12), 1099-1103 (2010).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+and+modularity+in+cell-fate+decision+making.">Memory and modularity in cell-fate decision making.</a> Nature. 503 (7477), 481-486 (2013).</li><li>Perestrelo, A. R., Aguas, A. C., Rainer, A., Forte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Organ/Body-on-a-Chip+Devices+at+the+Convergence+of+Biology+and+Microengineering.">Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and Microengineering.</a> Sensors. 15 (12), 31142-31170 (2015).</li><li>Johari, S., Nock, V., Alkaisi, M. M., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+analysis+of+C.+elegans+muscular+forces+and+locomotion+patterns+in+microstructured+environments.">On-chip analysis of C. elegans muscular forces and locomotion patterns in microstructured environments.</a> Lab Chip. 13 (9), 1699-1707 (2013).</li><li>Liu, J., 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=Coupling+between+distant+biofilms+and+emergence+of+nutrient+time-sharing.">Coupling between distant biofilms and emergence of nutrient time-sharing.</a> Science. 356 (6338), 638-642 (2017).</li><li>Choi, C. H., 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=One-step+generation+of+cell-laden+microgels+using+double+emulsion+drops+with+a+sacrificial+ultra-thin+oil+shell.">One-step generation of cell-laden microgels using double emulsion drops with a sacrificial ultra-thin oil shell.</a> Lab Chip. 16 (9), 1549-1555 (2016).</li><li>Weibel, D. B., Diluzio, W. R., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication+meets+microbiology.">Microfabrication meets microbiology.</a> Nat Rev Microbiol. 5 (3), 209-218 (2007).</li><li>Taheri-Araghi, S., Brown, S. D., Sauls, J. T., McIntosh, D. B., Jun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Physiology.">Single-Cell Physiology.</a> Annu Rev Biophys. 44, 123-142 (2015).</li><li>Karimi, A., Karig, D., Kumar, A., Ardekani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+physical+mechanisms+and+biofilm+processes:+review+of+microfluidic+methods.">Interplay of physical mechanisms and biofilm processes: review of microfluidic methods.</a> Lab Chip. 15 (1), 23-42 (2015).</li><li>Yu, F. 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. K., Shen, Y., Stone, H. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization,+competition,+and+dispersal+of+pathogens+in+fluid+flow+networks.">Colonization, competition, and dispersal of pathogens in fluid flow networks.</a> Curr Biol. 25 (9), 1201-1207 (2015).</li><li>Connell, J. 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. A., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+in+a+phosphorelay+drives+stochastic+entry+into+sporulation+in+Bacillus+subtilis.">Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis.</a> EMBO J. 36 (19), 2856-2869 (2017).</li><li>Cabeen, M. T., Russell, J. R., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+microfluidic+platform+to+uncover+basic+features+of+energy+and+environmental+stress+responses+in+individual+cells+of+Bacillus+subtilis.">Use of a microfluidic platform to uncover basic features of energy and environmental stress responses in individual cells of Bacillus subtilis.</a> PLoS Genet. 13 (7), e1006901 (2017).</li></ol>

作者没有什么可透露的。

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. 枯草细胞的养分和代谢物的流量, 特别是在长 (> 75 µm) 海沟5;但是, 单层功能通常足以满足最佳的细胞生长, 通常用于大肠杆菌, 特别是当它们较短 (约25µm)4。我们通常使用更长的战壕, 因为它们减少了随机出现的B. 枯草细胞链的频率, 它们被在主喂养通道5中的介质流拉出它们的沟槽。
进一步的修改, 如使用具有不同特征尺寸的设备 (例如,如、通道宽度) 或特征 (如、形状、开放端数) 可以适当地替换.例如, 在其他研究1、20、21中使用了在两端打开的单元格沟槽 (而不是在本协议的一端)。两端开放的战壕避免了细胞老化, 因为它们不断地充满新生细胞 (相对于母体机器, 最古老的细胞被跟踪, 新的细胞被推出通道), 虽然老细胞被推挤的事实两端通常将其观测窗口限制为少于10代1,21。我们通常寻求由一台母机器提供的较长的观测窗口 (数百代), 这使得有可能测量甚至记忆的进程, 等待时间是数万或数百代长的5。我们还使用了母机器设计来观察不呈指数增长的细胞, 如我们对产22的分析。在这种情况下, 可以对整个生长沟槽中的额外细胞进行有意义的分析22。
《议定书》中的许多步骤都是相对宽容的, 因为它们可以在不影响议定书结果的情况下加以修改。例如, 一个早期版本的协议要求清洁的盖子玻璃的顺序10分钟浴缸超声步骤在10米 KOH, 然后纯净水, 但我们发现, 良好的设备结合和操作是通过更简单的异丙醇基这里描述的清洁步骤。如果出现了粘接问题, 引起怀疑的清洁盖玻璃, 恢复到一个更严格的清洁协议建议。同样, 当我们使用离心步骤来最大限度地增加细胞的沟槽负荷, 即使没有这一步, 也可以达到合理的全负荷, 只要将非常集中的细胞用于加载步骤。对于没有现成的离心机适配器来旋转设备的研究人员来说, 这种替代策略尤其有用。此外, 可以使用不同浓度的钝化/润滑剂 (BSA 在本议定书);我们经常使用浓度高达1毫克/毫升。如果一个菌株可能对 BSA 敏感, 即使在没有钝化剂的情况下也可以取得可接受的结果。事实上, 当我们使用微流体设备检查产, 这需要细胞饥饿, 我们担心 BSA 可能作为一个潜在的营养来源的细胞, 所以我们省略了它从介质流没有观察到任何实质性不利影响22。
值得注意的是, 通过该装置的连续介质流动, 与封闭系统 (如烧瓶) 中的细胞生长相比, 可引出稍有不同的表型。例如, 营养或复合损耗可能具有挑战性, 因为细胞通常会从介质流动中清除微量化合物。事实上, 我们已经观察到, 通过饥饿诱导细胞产比在一个烧瓶中需要更长的时间在微流控装置。同样, 我们观察到, 利用氧化磷酸化器羰基氰基腙 (CCCP) 的能量应力诱导, 微流控装置中的多倍高浓度比在烧瓶培养中报告的多。23. 因此, 在不同的介质添加剂下, 可能需要进行一些优化, 以达到满意的效果。

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><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>(240)4019862</td><td>This is referred to as PDMS in the protocol. There are 2 components that are mixed at a 10:1 ratio</td></tr><tr><td>0.75-mm biopsy punch</td><td>World Precision Instruments</td><td>504529</td><td></td></tr><tr><td>22 x 40 mm No. 1.5 cover glass</td><td>VWR</td><td>48393 172</td><td></td></tr><tr><td>Plasma Etch PE-50</td><td>Plasma Etch Inc.</td><td>PE-50</td><td>Instrument used to bond PDMS to glass using oxygen plasma treatment</td></tr><tr><td>Tygon flexible tubing ID 0.02&quot;, OD 0.06&quot;</td><td>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><td>Silicone Tubing, 0.04&quot; ID, 0.085&quot; OD</td><td>HelixMark Standard Silicone Tubing</td><td>60-795-05</td><td>For pinch valves and Y-junction connection</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-100G</td><td>Used as passivation agent</td></tr><tr><td>ART Gel Pipet Tips (P200)</td><td>Molecular BioProducts</td><td>2155</td><td>For loading the device with medium and cells</td></tr><tr><td>Acrodisc 32-mm syringe filter with 5-&amp;mu;m Supor membrane</td><td>Pall Life Sciences</td><td>4560</td><td>For filtering cultures before device loading</td></tr><tr><td>21-ga blunt needles, 1&quot;</td><td>McMaster-Carr</td><td>75165A681</td><td></td></tr><tr><td>Y connector with 200-series barbs, 1/16&quot; ID tubing, polypropylene</td><td>Nordson Medical</td><td>Y210-6005</td><td>The company is AKA Value Plastics</td></tr><tr><td>Eclipse Ti-E inverted microscope</td><td>Nikon</td><td></td><td>This unit has been discontinued by the manufacturer and is replaced by the Ti 2 E.</td></tr><tr><td>LB Lennox</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>Microfuge 18</td><td>Beckman Coulter</td><td>367160</td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; strain NCIB3610</td><td>Bacillus Genetic Stock Center</td><td>3A1</td><td>wild-type parental strain modified for use in the experiments shown in this protocol</td></tr><tr><td>Gravity convection oven</td><td>VWR</td><td>414005-106</td><td>for curing PDMS and baking assembled devices</td></tr><tr><td>Scotch-Weld Epoxy Kit</td><td>3M</td><td>2216 B/A</td><td>may be used to bond a silicon wafer to an aluminum backing plate</td></tr><tr><td>Scotch Magic tape, 3/4&quot; width</td><td>3M</td><td></td><td>to clean dust from PDMS devices and to place over features to increase visibility (denoted &quot;office tape&quot; or &quot;adhesive tape&quot; in protocol)</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ800N</td><td>listed here as an example; nearly any stereomicroscope will do</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td>W292907</td><td>To clean cover glass</td></tr><tr><td>Syring pump, 6 channel</td><td>New Era Pump Systems Inc.</td><td>NE-1600</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>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, 可以使用, 但结果数据密度将较低, 由于较少的细胞谱系被成像在每个影像的位置。
一旦设备最初加载, 它是平衡通过连接微流体管道和流动介质通过设备, 在一个相对高的流量为35µL/分钟。平衡的步骤, 以消除气泡和过剩的细胞在喂食通道从设备, 并鼓励细胞在侧通道开始增长。在介质流动时从设备上移除的细胞通常可以被肉眼观察到, 它是通过出口导管向废物容器移动的光色带;这种波段的运动是一个视觉指示器, 流体通过管道和装置正常流动。对平衡装置的显微检查应能在大多数侧通道 (图 3B, 0 分钟) 内显示单个文件中的少数单元格。
一旦平衡装置被放置在显微镜动在 1 . 5 µL / 分钟在37° c , 细胞将恢复均匀和恒定的指数相增长的过程中约 2 小时 (图 3B) 。在细胞的生成时间内, 在图像分析后, 应通过高原的出现, 直接验证恒定的指数增长。在这一点上, 细胞已准备好荧光和/或明成像的期望。成功加载和平衡的设备通常可以可靠地映像至少24小时 (图 4A, C)。引入一个中型交换机 (图 4A-C) 扩展了可以进行的实验范围, 但也增加了灾难性细胞死亡事件的频率 (图 4D), 大概是通过引入空气未成功从油管中清除的气泡。另一个可能导致灾难性细胞死亡的事件是, 位于入口的细胞簇可能会脱落并通过进给通道, 如图 4D所示。我们目前还不明白为什么这会导致设备中的灾难性细胞死亡, 我们还没有设计出一种方法来防止这种事件的发生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56901/56901fig1.jpg"> 图 1: 微流控装置示意图设备示意图大致显示为缩放。字母指示符与入口和出口区域一起被标记, 被猛击连接钝器结束的针。通常, 使用一半的图案通道 (灰色阴影)。细胞海沟朝向指示符方向。随附的 CAD 文件 (请参见补充文件 1) 显示了设备上的所有功能。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56901/56901fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56901/56901fig2.jpg"> 图 2: 微流体设备的钝化、单元加载和平衡.(a) 在含有 BSA 介质的钝化后的粘合装置。在出口孔中保存有凝胶加载的小贴士, 通过该装置监测介质和细胞的通道。中等半月板是可看见的在稀薄的部分胶凝体装货技巧 (箭头)。(B) 单元格加载后的设备。在凝胶加载提示 (箭头) 中, 细胞可见为半透明的白色层。(C) 在离心前在特制离心式适配器中除去凝胶加载提示。医用胶带 (蓝色) 放置在铝件上以缓冲装置。(D) 在进行离心和加载验证之后, 该装置被贴在显微镜下, 插入并附着在入口和出口引脚上。在所示的图像中, 由夹紧阀和 Y 连接器组成的介质开关是由微尖端盒中的装置所设置的。(E) 整个组装装置的示意图, 从注射器泵到废物容器。出口油管运行到一个小的废物烧杯。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56901/56901fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56901/56901fig3.jpg"> 图 3: 微流控设备中的电池加载和增长.(a) 从左到右, 在单元加载前只含有介质的装置的相位对比度显微, 在离心后, 通过移, 但在离心力作用下, 同一个装置, 在离心泵后加入相同的装置。(B) 单元格适应和增长的时间过程 (LB, 37 ° c, 流量1.5 µL/分)。在适应初期, 固定相细胞相对较短和狭窄。随着细胞的适应和恢复到指数相增长 (60-120 分钟), 他们采取了更长和更广泛的形态学。120分钟后, 设备中的所有单元都恢复了一致的指数相位增长, 并且设备已准备好进行成像。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56901/56901fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56901/56901fig4.jpg"> 图 4: 微流控设备中的细胞生长和介质切换.(a) Kymographs 显示550分钟的生长前和1000分钟的增长后, 一个中型交换机 (灰色虚线) 为2% 乙醇作为一个压力源。图像以10分钟的间隔捕获。顶部的面板显示了一个转录 mNeonGreen 记者 (绿色通道) 的压力诱导基因。底部面板显示了本例中用于自动细胞分割的本构 mNeptune 报告器 (red 通道)。(B) 在面板 A 的虚线框中 kymographs 部分的关闭视图, 显示在介质开关之后的绿色通道中的瞬态响应。注意, 尽管乙醇的存在会使细胞变得更短, 并以稍慢的速度生长, 但这种转变仍在继续。时间刻度由水平条显示, 距离由竖线显示。(C) 示例从面板 a 中所示的实验中分析单个细胞谱系的荧光数据的痕迹。垂直虚线表示介质开关为2% 乙醇作为压力源。(D) 失败的中型交换机的示例。当第二介质到达单元格时, 通道中的单元格裂解。该开关是伴随着大量的细胞, 通过在主通道, 造成一个明亮的荧光信号 (与冲出区域相对应的再缩放图像显示在它上面)。在开关后, 没有进一步的细胞被观察, 虽然微弱的萤光细胞残骸遗骸的在渠道。时间刻度由水平条显示, 距离由竖线显示。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56901/56901fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

single-cell-microfluidic-analysis-of-bacillus-subtilis

Nanyang Technological University

Research

JoVE Journal

Biology

Single-cell Microfluidic Analysis of Bacillus subtilis

11.9K 次观看.  Oklahoma State University. 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。

视频: Single-cell Microfluidic Analysis of Bacillus subtilis

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

生物工程

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

环境科学

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

生物学

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

生物化学

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...