Name: ecosystem fabrication (ecofab) protocols for the construction of laboratory ecosystems designed to study plant-microbe interactions
Uploaded: 2018-04-10T13:30:00.000+00:00
Duration: 11 min 57 s
Description: Watch this Scientific Journal Video about Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions at JoVE.com

这项工作得到了劳伦斯伯克利国家实验室的实验室指导研究和开发 (LDRD) 计划的支持, 由美国能源部根据合同编号支持。DE-AC02-05CH11231 和 DE-SC0014079 由美国能源部科学办公室授予加州大学伯克利分校。在美国能源部的合同号下, 分子铸造厂的工作得到了支持。DE-AC02-05CH11231。我们还感谢苏珊娜 m. Kosina, 凯瑟琳路易, 本杰明 p. 伯恩, 以及在劳伦斯伯克利国家实验室的本杰明 j. 科尔的所有帮助。

注意: 本议定书包括使用危险化学品、锋利物体、电器设备、热物体以及可能造成伤害的其他危险。适当的个人防护设备 (PPE、 e. g、耐化学性手套、安全眼镜、实验室大衣、长衣服、闭趾鞋、等) 应佩戴, 并适当的安全程序 (安全培训, 使用油烟机,等。应遵循。
1. EcoFAB 器件制造: 铸造微结构层 (图 2 &图 3)
<ol><li>使用3D 打印技术构造 EcoFAB 模具 (可在中提供设计文件。每个模具包括三部分: 铸造框架、特色模具底座和插入, 如图 2所示。使用3D 塑料打印机打印模具底座并插入刚性不透明 photopolymers。利用最小100µm 分辨率, 用丙烯腈丁二烯苯乙烯 (ABS) 打印铸件框架。</li>
	<li>在一次性的1升容器中, 将40毫升的硅氧基弹性体与固化剂混合使用。根据所需的试验 (步骤2.1 和 2.2), 使用不同比例 (v/v) 的弹性体基础/固化剂 (5:1, 15:1, 或 30:1)。对各种混合物进行步骤1.3 至1.8。 注意事项: 佩戴耐化学性的手套、安全眼镜和其他 PPEs。</li>
	<li>将容器放在真空室中至少30分钟, 从弹性体混合物中除去气泡。</li>
	<li>将混合物倒入组装的3D 印刷塑料模具 (图 3A) 中, 并将模具放在加热块上, 温度为85摄氏度, 为 4 h (图 3B)。 注意: 佩戴防护用品以避免烧伤。</li>
	<li>让模具冷却5分钟。然后轻轻拉出模具中的插入 (图 3C), 然后在铸件框架和 (固化的弹性体混合物) 之间慢慢插入一把实用刀, 将它们分开 (图 2D)。</li>
	<li>在铸件框架 (图 3E) 上, 按模具底座。使用刀或其他工具轻轻地将所用的注塑层与边缘的模具底座 (图 3F) 分开, 然后将其从模具表面慢慢剥离 (图 3G)。</li>
	<li>通过用15口径钝针 (图 3H, I) 为入口和出口端口制作〜1.6 毫米孔, 在该层上创建入口和出口通道。 注: 标准模具有入口和出口端口, 而宽出口模具只需要入口端口 (图 3H, I)。</li>
	<li>使用剪刀修剪该层的边缘. 注: 修剪后的≥76层应为小型 EcoFAB 设备和≥102毫米 x 83 毫米矩形的大型 EcoFAB 设备的 mm x 51 毫米矩形。</li>
</ol>2. EcoFAB 器件制造: 化学附着在显微镜上的玻片 (图 3 &图 4)
<ol><li>永久性地将其层与显微镜幻灯片结合在一起<ol>
<li>冲洗的结合面 (由15:1 弹性体基础, 以固化剂混合物) 和 7.6 x 5 厘米显微镜幻灯片与甲醇, 然后吹干的压缩空气或超纯氮气枪。 注意: 甲醇是有毒的。工作在油烟机和佩戴防护眼罩, 手套和其他 PPEs。</li>
		<li>将显微镜滑块和玻纤层置于等离子清洗器中, 其粘接面朝上 (图 3J)。如果等离子清洗器不可用, 请跳至步骤2.2。</li>
		<li>关闭等离子清洗器的腔室和排气阀, 打开真空, 并在腔内泵入1分钟。</li>
		<li>打开等离子发生器的电源, 将射频 (RF) 电平转换为 "HI" 1 分钟。</li>
		<li>关闭真空泵和等离子电源, 并将燃烧室排出大气层。</li>
		<li>从等离子腔中取出显微镜滑块和玻纤层, 并在均匀压力的情况下, 快速按下四边缘的聚硅烷层到幻灯片上 (图 3L)。确保该层的中心椭圆形区域 (根腔) 不接触幻灯片。</li>
		<li>将密封的 EcoFAB 装置放在120摄氏度的加热块上20分钟, 进一步保证了该层与显微镜滑动之间的永久性粘合。</li>
		<li>让设备冷却5分钟, 用小刀修剪一层的额外边缘。</li>
	</ol>
</li>
	<li>玻片的可逆物理密封<ol>
<li>可逆密封技术采用了一套自定义夹具 (由3D 塑料打印机打印或用金属加工, 绘图显示在图 4中)。<ol>
<li>将显微镜滑入底部钳板上的切口上, 然后将该玻片层 (由5:1 弹性体底座制成的固化剂混合物) 对准幻灯片顶部。</li>
			<li>将顶部钳板放在该层上。使用四个六角帽螺钉将顶部和底部板固定在一起, 将螺钉定向, 使螺母从夹具顶部螺纹。</li>
		</ol>
</li>
		<li>直接附着在显微镜幻灯片上<ol>
<li>在显微镜下滑动的顶部放置一层 (由30:1 个弹性体底座制成的固化剂混合物)。</li>
			<li>在幻灯片上按下一层。软的, 非常粘接的 30:1) 应坚持幻灯片创建一个防水密封, 没有永久性的化学键或物理压力从夹具 (图 3L)。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. EcoFABs 杀菌
<ol><li>用超纯水冲洗 EcoFAB 装置。</li>
	<li>将一个 EcoFAB 设备放在 EcoFAB 容器中, 然后添加70% 乙醇, 直到设备被淹没。关闭容器盖, 轻轻摇动, 将所有表面与乙醇混合。确保 EcoFAB 装置的根生长室充满乙醇, 很少或没有气泡。</li>
	<li>在室温下孵化30分钟后, 倒入70% 乙醇, 用100% 乙醇重复孵化5分钟。</li>
	<li>排出乙醇, 在层流罩中孵化16小时的灭菌 EcoFAB, 使其完全干燥。如果可用, 通过打开罩内的 UV 光1小时消毒系统。 注意: 使用紫外线灯时应佩戴适当的 PPE。</li>
	<li>将灭菌后的 EcoFABs 存放在消毒罩或蒸气袋中, 供日后使用。</li>
</ol>4. 带 LED 长光源的 EcoFABs (图 5)
<ol><li>将 LED 带连接到 EcoFAB 容器上<ol>
<li>将 EcoFAB 容器上的位置标记为9个 LED 剪辑。从容器底部沿边缘 (图 5A) 开始, 第一个剪辑 120 mm, 然后继续在容器周围的螺旋标记出剪辑位置, 每个下一个剪辑将下降 10 mm。9个剪辑的螺旋, 允许1米的 LED 带环绕容器两次。</li>
		<li>通过将两个热胶戴博斯到容器上, 将 LED 剪辑粘附到每个标记位置, 并与剪辑的安装孔位置对齐。将夹孔压入这两戴博斯胶水中, 然后在孔的顶部添加另一张胶水。对所有剪辑重复该过程, 直到9个剪辑形成螺旋形 (图 5B)。 注意: 使用热胶水时要戴上手套和其他 PPE 以避免烫伤。</li>
		<li>将 led 条螺纹通过螺旋形状的剪辑, 并将 led 朝向容器。该条应绕两次循环 (图 5C)。</li>
	</ol>
</li>
	<li>使用控制器连接 LED 带到电源 (图 5D显示带有照明灯的 EcoFAB 室, 控制器的编程在步骤4.3 中描述)。 注意: 电击危险: 在连接电线时要确保电源被拔掉。<ol>
<li>将电源的正负端子连接到 "输入: v +" 和 "输入: v-" 控制器的终端, 使用2线缆 (图 5E显示控制器设置的示意图图)。</li>
		<li>将负引线从 "母到裸" 电缆的裸端连接到控制器上的一个 "输出" 通道。 注: 该协议中使用的控制器有五个通道, 因此可以支持多达五1米的 LED 带。</li>
		<li>将缆线的所有正引线连接到紧凑拼接连接器 (如果需要多个通道), 然后将此连接器链接到控制器的 "输出 V +" 终端。</li>
		<li>将每个 led 带插入到电缆的母端, 这样每个 led 都有自己的通道来控制。如果需要, 使用女性对男性电缆延伸的范围。</li>
	</ol>
</li>
	<li>根据制造商的指示, 为所需的光周期编程控制器,</li>
</ol>5. EcoFABs 种植植物
<ol><li>种子杀菌与萌发 注: 种子灭菌和所有后续步骤与幼苗必须在无菌条件下进行。下面讨论的灭菌过程适用于拟南芥、阿韦纳莲、Brachypodium distachyon和柳枝柳枝稷的种子。柳枝柳枝稷种子在灭菌过程前应在60% 硫酸中悬浮1小时。建议 1-2 种子每 EcoFAB 装置, 考虑发芽率和均匀性的发芽。<ol>
<li>将种子浸泡在70% 乙醇中2分钟。</li>
		<li>用吸管除去乙醇, 用无菌水冲洗种子三次。</li>
		<li>将种子留在10% 漂白剂溶液中5分钟。</li>
		<li>除去漂白液, 用无菌水彻底清洗种子三次。</li>
		<li>在种子中加入无菌水, 在4摄氏度的冰箱中孵化出离心管7天。</li>
		<li>将种子均匀地分布在 0.5 Murashige & Skoog (MS) 培养基上, 0.6% phytagel, 用微孔胶带封住板材。</li>
		<li>将植株长为大约5毫米的根长度, 以便传输到 EcoFABs (图 6A)。在这里提出的实验, 在22°c 生长室应用一个 16 h light/8 h 黑暗照明的制度, 并孵化植物 2-7 d 前转移到 EcoFAB (2 天为阿韦纳莲和Brachypodium distachyon, 7 天为拟南芥拟南芥和柳枝柳枝稷)。</li>
	</ol>
</li>
	<li>用液体介质将幼苗转移到 EcoFABs (图 6)<ol>
<li>使用无菌注射器或微, 用无菌水冲洗 EcoFAB 设备的根腔三次, 然后用感兴趣的生长介质填充根腔, 例如 0.5 MS 介质 (图 6B, 步骤 5.1.6)。</li>
		<li>小心地将单个幼苗插入 EcoFAB 设备的植物库中 (图 6C)。 注: 根应完全浸没在根腔内, 并将其射出储层。</li>
		<li>在容器中加入3毫升的无菌水, 避免 EcoFAB 装置。这将增加湿气和减少介质从根室蒸发。</li>
		<li>关闭容器, 用微孔胶带封住盖子 (图 6D)。</li>
		<li>将 EcoFAB 放入植物孵化器, 或利用 EcoFAB 照明系统, 在一个适合于各自植物生长的受控温度环境中 (步骤 4)。为这个研究, 设置房间到24°c。</li>
		<li>定期检查 EcoFAB 在根生长室内重新填充生长介质, 并将水添加到容器中。在无菌条件下执行所有步骤。 注意: 对于早期的植物生长阶段, 每5到7天就需要重填根系生长室。对于后期的生长阶段, 每2到3天就需要再加注一次。如果需要, 使用注射器或吸管从根生长室收集根渗出液到离心管中, 并将其贮存在-80 摄氏度冷藏库中;此外, 图像的根形态学与凝胶成像或显微镜。</li>
	</ol>
</li>
	<li>用固体基质将幼苗转移到 EcoFABs 中<ol>
<li>如果使用一组自定义夹具将其连接到显微镜幻灯片 (图 3K,图 4), 则使用用5:1 混合弹性体底座制造的根腔来固化代理。或者选择一个由30:1 碱基组成的, 以固化剂混合物为基础的一层, 如果直接将其粘附到幻灯片上 (如步骤2.2 所述)。</li>
		<li>消毒 EcoFAB 室, 如步骤3所述。</li>
		<li>小心地将被灭菌的土壤/沙子添加到根腔中, 将基材层倒置, 并将基体添加到根腔中。避免在与显微镜滑动接触的区域内的任何微粒掉落, 因为这将减少附着力。</li>
		<li>将显微镜幻灯片放在该层的顶部, 并用力压紧所有的边缘。小心翻转 EcoFAB 装置, 使土壤/沙子从油藏开口中脱落。 注: 对于 EcoFAB 设备的5:1 碱固化剂混合物, 使用自定义夹具, 以确保密封。</li>
		<li>通过 EcoFAB 装置的入口或出口通道流动液体介质或水, 并将幼苗转移到其植物油藏中, 如步骤3.3 所述。</li>
	</ol>
</li>
	<li>将微生物添加到 EcoFABs 中<ol>
<li>将微生物菌落转移到具有8毫升 LB 汤的孵化管, 并将其生长到 OD 0.5 (约12小时)。</li>
		<li>将培养液转化为15毫升离心管, 在室温下将其离心为5分钟, 在 3000 x g 到颗粒微生物。</li>
		<li>移除上清液, 并添加8毫升的植物生长培养基用于靶 EcoFAB。悬浮颗粒的微生物, 并离心管在室温下5分钟在 3000 x g。</li>
		<li>重复步骤5.4.3。两次完全清除任何 LB 营养痕迹。</li>
		<li>将植物生长培养基添加到洗涤过的微生物颗粒中, 直到其光密度约为 600 nm 的0.5。</li>
		<li>通过 EcoFAB 出口将微生物溶液的20µL 添加到根室中。本出版物中使用的菌株在 2-3 天内进行植物根系的种植, 并开始对根表面进行殖民。</li>
		<li>对于工程化学发光, 确保包括诱导剂 (1 毫米 IPTG) 在植物生长培养基诱导荧光素酶的表达。</li>
	</ol>
</li>
</ol>6. EcoFABs 根分泌物的代谢产物分析
<ol><li>基于新陈代谢分析的 LC/MS 样品制备方法研究<ol>
<li>将离心管从 EcoFABs 中收集到的根分泌物从冻干机中取出, 并打开冻干机以除去管子中的所有水。</li>
		<li>将 LC-MS 级甲醇的300µL 加入每管, 油脂实验30分钟。 注意: 使用甲醇时要佩戴 PPE。</li>
		<li>把管子放在离心机里, 在 3000 x g 处离心5分钟。</li>
		<li>将上清溶液转化为新的离心管, 在真空浓缩器中蒸发甲醇。</li>
		<li>将150µL 甲醇与1毫米 LC-MS 内部标准放入每管, 并在4°c 冰箱中孵化出12小时的管。</li>
		<li>离心管在 3000 x g 5 分钟, 并转移到上清到0.22 µm 过滤管。</li>
		<li>离心过滤管, 并将过滤后的溶液转化为2.0 毫升 LC/MS 瓶, 200 µL 插入。</li>
		<li>将瓶子放在 lc/ms 机架内, 并将机架装入 lc/ms 自动取样器内。</li>
	</ol>
</li>
	<li>数据分析<ol>
<li>获取代谢物图谱和自定义 Python 脚本26或使用其他数据分析软件的访问。</li>
		<li>使用代谢物标准库确定基于 m/z值、保留时间和复合碎片模式的代谢产物。27
</li>
	</ol>
</li>
</ol>7. EcoFABs 植物根的质谱成像 (图 7)
注: 用自定义夹具 (图 7A) 将5:1 弹性体底座制成的 EcoFAB 装置用于将根冲压到纳米结构引发剂质谱 (随着 nims) 芯片上, 28, 29, 30因为可反向粘贴到随着 nims 芯片的表面。
<ol><li>用 UV 光随着 nims 芯片表面消毒1小时。</li>
	<li>从孵化器中挑选一株生长植物的 EcoFAB, 把它放在消毒罩里。</li>
	<li>打开 EcoFAB 容器, 卸下夹顶板。</li>
	<li>与内部的工厂一起提起, 并小心地将其与工厂连接在随着 nims 芯片上 (图7B、D、E)。 注: 一旦根接触随着 nims 芯片表面, 不应移动。这样可以防止根代谢物的 "涂抹"。</li>
	<li>轻轻地按下的根, 通过该层, 直到根完全接触随着 nims 表面。把根放在随着 nims 表面上20分钟。</li>
	<li>从随着 nims 芯片上提起包括该植物的该层, 再次避免在随着 nims 表面移动根。如果需要, 将工厂退回到夹具。</li>
	<li>将随着 nims 芯片连接到自定义 MALDI 板上, 并将该板加载到 MALDI 光谱仪中以进行质量成像 (图7C)。</li>
	<li>使用 OpenMSI 程序生成根代谢物的随着 nims 图像 (图7D-G) 31.</li>
</ol>

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作者没有什么可透露的。

在这里报告的使用生态系统制造创建 EcoFABs 提供社区资源的系统植物生物学研究在高度控制的实验室条件。3D 印刷技术的进步为构建和迭代精炼 EcoFAB 设计提供了广泛的方便。这里提出的根室是非常适合的成像显微镜和维持不育, 使受控添加微生物, 以调查植物微生物相互作用。EcoFAB 平台与各种植物品种相容。重要的是要认识到生长在狭窄的根腔内的植物的生理效应, 这样就需要额外的实验来将发现推广到在自然环境中生长的植物。
使用无菌室和 LED 生长灯, 可以调查各种光照条件的影响, 包括波长, 强度和持续时间, 对植物生长和相关的生理参数的平行。可逆的粘合根室允许使用固体基底以及空间收集固体样品进行生化和遗传分析。固体基质 (如土壤、沙子和石英珠) 的应用提供了利用 EcoFABs 构建更具生态学意义的实验室生态系统的可能性。然而, 这里提出的所有系统都使用饱和液体 (水培栽培), 这是不准确的反映大多数土壤, 这将是重要的是进一步细化这些设计, 以保持空气袋在土壤中, 使他们更好地代表天然土壤。
简单的照相机和显微镜的使用被描述对映像根系统形态学发展在大到细胞水平。这种适合的监测根形态学成像和定量将有助于了解植物生理和分子信号触发的植物基因型 adaptions 的生长条件的调控机制。然而, 研究生理根发育的一个局限是 EcoFAB 装置的当前水平位置。在自然环境中, 根 gravitropic 反应导致根系统的主要垂直发展。因此, 这里所呈现的水平系统在某些因素中可能不同于自然环境, 而 EcoFAB 系统的制作, 垂直放置的根腔是未来 EcoFAB 版本的理想目标。虽然目前的 EcoFAB 设备是水平放置, 分析的根形态参数在不同的条件, 或对微生物的反应, 是可能的。高分辨率成像可用于捕获单个孤立物或群落的根殖民化动力学, 提供有关哪些植物部分在各种养分充足和不足条件下被殖民的信息。预计这类研究将为植物微生物的组装提供重要的新见解, 以及这些动态如何随着时间的推移而变化, 例如根系的发展。
微流控装置使非常年轻的植物成像, 通常收集的代谢物的数量是不够的 LCMS 分析。土壤为基础的系统, 如 rhizotrons, 允许在植物转化为化学发光结构 (格洛根) 或以核磁共振为基础的方法33,34时, 根形态学的成像。由于大量的样品, 从这些系统中提取代谢物是费时费力的。EcoFABs 是两者的结合: 制造类似于微流控设备。EcoFABs 的设计是简单和廉价的繁殖, 但房间的大小可以调整, 以种植小的或大的根系系统, 直至其生殖阶段。同时观察根形态学改变和根渗出是可能的。该系统是无菌的, 能够控制特定微生物的添加。
EcoFABs 的设计是为了控制微生物和代谢物的导入和取样。具体地说, 从根生长室收集的样本被发现足以用于质谱代谢物的分析。质谱成像的集成 (随着 nims) 提供了一种非破坏性的方法来研究根系系统的代谢物空间分布.该技术可能有助于今后稳定同位素追踪实验, 并将微生物定位映射到特定代谢物36。虽然本议定书侧重于单一隔离, 但同样的设计也可以用于更复杂的社区。EcoFABs 中的样本量和生物量很可能足以与 DNA 测序技术进一步融合, 这对于表征和监测微生物群落结构和基因表达具有重要意义。
最后, 本议定书详述了为调查植物-微生物相互作用而设计的实验室生态系统的制作, 重点放在简单和容易接近的方法上, 研究人员可以很容易地实施和扩展世界。目前的努力旨在证明实验室之间的重复性和温度控制系统的集成, 使每个 EcoFAB 将有独立控制的光和温度。该系统的进一步发展将是整合 EcoFAB 根室的自动取样和再灌装, 并为在 EcoFABs 内建立相关的植物微生物制定可重现的协议。

有益植物微生物在农业中的应用提供了巨大的潜力, 以增加可持续的食品和生物燃料的生产, 以提供增长的人口1,2,3,4。大量的工作支持植物微生物在植物养分摄取、耐应力和抗病性方面的重要性5,6,7,8。然而, 由于复杂性和相关 irreproducibility, 无法精确控制微生物组成和遗传学 (e. g), 因此很难对田间生态系统中植物-微生物相互作用的机制进行调查。微生物突变体)4,9,10。
一个战略是建立简化的模型生态系统, 使受控制的, 复制的实验室实验, 调查植物-微生物相互作用, 以产生洞察力, 可以进一步测试在领域10,11, 12。这一概念建立在传统的方法上, 使用种植在土壤填充盆或在温室或孵化器内的琼脂板的植物13。虽然这些可能仍然是最广泛使用的方法, 但它们缺乏精确监测和操作植物生长环境的能力。为了达到这些目的, rhizoboxes 和 rhizotrons 代表了研究以下地面进程的能力的重大改进14,15, 并发布了第一个协议来分析土壤中的根际代谢产物16。最近, 为了启用高通量分析, 高级微流控设备13,17 , 如工厂芯片18,19, RootArray20和 RootChip21, 已开发为植物分型的有效工具, 用微米尺度空间分辨率来监测小模型植物拟南芥在液体流动培养基中的早期生长阶段。最近, 介绍了一种双层成像平台, 它能使拟南芥在幼苗阶段的根发成像具有微流控平台22。
这里提供了构建受控实验室设备 (EcoFABs) 的详细协议, 用于研究植物微生物相互作用, 并表明它们可用于研究各种植物, 包括拟南芥、 Brachypodiumdistachyon23, 生态上重要的野生燕麦阿韦纳莲、和生物能源作物柳枝柳枝稷 (柳枝稷)。EcoFAB 是一种不育植物生长平台, 包括两个主要成分: EcoFAB 装置和无菌植物大小透明容器。该 EcoFAB 设备是由一个烷的制造过程, 包括从3D 印刷塑料模具铸造和结合在显微镜幻灯片上, 使用以前报告的方法2425.本协议 (图 1) 描述了 EcoFAB 工作流的详细程序, 如设备制造、杀菌、种子萌发、苗移植、微生物接种/cocultivation、样品制备和分析。对基本工作流的进一步修改进行了描述, 包括安装计算机控制的 LED 生长灯和固体基板的使用。介绍了影像学技术在研究根系形态变化、根系微生物定植、根系分泌物质谱成像等方面的应用。我们预计, 基于现成材料的简单、廉价的设计, 以及此处提出的详细协议, 将使 EcoFAB 平台成为社区资源, 规范实验室植物微生物学研究。

<table><tbody><tr><td>3D printed custom mold</td><td>LBNL</td><td></td><td>STL files available here www.eco-fab.org; The EcoFABs molds described here were printed by FATHOM: http://studiofathom.com</td></tr><tr><td>Dow sylgard 184 silicone elastomer clear kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>Air duster spray</td><td>VWR</td><td>75780-350</td><td>any compressed gas duster should work</td></tr><tr><td>15 gauge blunt needle</td><td>VWR</td><td>89166-240</td><td></td></tr><tr><td>5 mL syringe with Luer-Lok Tip</td><td>VWR</td><td>BD309646</td><td></td></tr><tr><td>3&amp;rdquo;x2&amp;rdquo; microscope glass slide</td><td>VWR</td><td>48382-179</td><td></td></tr><tr><td>1.75&quot; x 2.56&quot; x 3.56&quot; EcoFAB box</td><td>Amazon</td><td>B005GAQ25Q</td><td></td></tr><tr><td>4&amp;rdquo; x 3 &amp;frac14;&amp;rdquo; microscope glass slide</td><td>Ted Pella</td><td>260231</td><td></td></tr><tr><td>4.87&quot; x 4.87&quot; x 5.50&quot; EcoFAB box</td><td>Amazon</td><td>B00P9QVOS2</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-001</td><td></td></tr><tr><td>3D printed custom clamp</td><td>LBNL</td><td></td><td>STL files available from Trent Northen&#039;s lab</td></tr><tr><td>Sterile hood</td><td>AirClean Systems</td><td>AC600 Series PCR Workstations</td><td></td></tr><tr><td>PTFE syringe tubing</td><td>Sigma-Aldrich</td><td>Z117315-1EA</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>89125-172</td><td></td></tr><tr><td>Bleach</td><td></td><td></td><td></td></tr><tr><td>Murashige and Skoog (MS) Macronutrient Salt Base</td><td>Phytotechnologies Laboratories</td><td>M502</td><td></td></tr><tr><td>Murashige and Skoog (MS) Micronutrient Salt Base</td><td>Phytotechnologies Laboratories</td><td>M554</td><td></td></tr><tr><td>Soil</td><td>Hummert International</td><td>Pro-Mix PGX</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>71010-52-1</td><td></td></tr><tr><td>Arabidopsis thaliana</td><td>Lehle Seeds</td><td>WT-24 Col-4 Columbia wild type</td><td></td></tr><tr><td>Brachypodium distachyon</td><td>LBNL</td><td>Standard Bd-21 line</td><td>Available from John Vogel&#039;s lab</td></tr><tr><td>Panicum virgatum</td><td>The Samuel Roberts Noble Foundation</td><td>Alamo switchgrass</td><td></td></tr><tr><td>Micropore tape</td><td>VWR</td><td>56222-182</td><td></td></tr><tr><td>LC-MS grade methanol</td><td>VWR</td><td>JT9830-3</td><td></td></tr><tr><td>Lyophilizer</td><td>LABCONCO</td><td>FreeZone 2.5 Plus</td><td></td></tr><tr><td>SpeedVAC concentrator</td><td>Thermo Scientific</td><td>Savant&amp;trade; SPD111 SpeedVac</td><td></td></tr><tr><td>Ultrafree-MC GV Centrifugal Filter-0.22 &amp;micro;m</td><td>Millipore</td><td>UFC30GV00</td><td></td></tr><tr><td>Liquid chromotography system</td><td>Agilent</td><td>Agilent 1290 LC system</td><td></td></tr><tr><td>Q Exactive mass spectrometer</td><td>Thermo Scientific</td><td>Q Exactive&amp;trade; Hybrid Quadrupole-Orbitrap MS</td><td></td></tr><tr><td>NIMS chip and custom MALDI plate</td><td>LBNL</td><td></td><td>For detailed protocol see: doi:10.1038/nprot.2008.110</td></tr><tr><td>MALDI mass spectrometer</td><td>AB Sciex</td><td>TOF/TOF 5800 MALDI MS</td><td></td></tr><tr><td>Nano-coated LED grow light strip</td><td>LED World Lighting</td><td>HH-SRB60F010-2835</td><td></td></tr><tr><td>Power supply</td><td>LED World Lighting</td><td>MD45W24VA, LV100-24N-UNV-J</td><td></td></tr><tr><td>TC420 controller</td><td>Amazon</td><td>B0197U7R8Q</td><td></td></tr><tr><td>Silicone LED clips</td><td>Amazon</td><td>B00N9X1GI0</td><td></td></tr><tr><td>Hot glue gun</td><td>Amazon</td><td>B006IY359K</td><td></td></tr><tr><td>Female-to-bare LED connector cable</td><td>LED World Lighting</td><td>HH-F05</td><td></td></tr><tr><td>Female-to-male LED connector extension cable</td><td>LED World Lighting</td><td>HH-MF1</td><td></td></tr><tr><td>20AWG 2-wire cable</td><td>LED World Lighting</td><td>6102051TFT4</td><td></td></tr><tr><td>WAGO 221-415 Splicing Connector</td><td>LED World Lighting</td><td>221-415</td><td></td></tr></tbody></table>

ecosystem fabrication (ecofab) protocols for the construction of laboratory ecosystems designed to study plant-microbe interactions

有益的植物-微生物相互作用提供了可持续的生物解决方案, 有潜力促进低投入的食品和生物能源生产。更好的机械理解这些复杂的植物-微生物相互作用将是至关重要的改善植物生产和执行基本生态研究, 调查植物-土壤-微生物相互作用。这里, 利用广泛可用的3D 印刷技术, 提出了生态系统制造的详细描述, 以建立受控实验室生境 (EcoFABs), 用于机械研究特定环境中的植物-微生物相互作用条件。描述了两种尺寸的 EcoFABs, 适合于与各种植物物种的微生物相互作用的调查, 包括拟南芥、 Brachypodium distachyon和柳枝柳枝稷。这些流动设备允许控制操作和抽样的根微生物, 根化学, 以及图像的根形态和微生物的定位。该协议包括在 EcoFABs 内保持无菌条件和将独立的 LED 光系统安装到 EcoFABs 上的详细信息。详细的方法, 以添加不同形式的介质, 包括土壤, 沙子, 和液体生长介质耦合的特点, 这些系统使用成像和新陈代谢描述。这些系统共同对植物和植物微生物联营集团进行动态和详细的调查, 包括对微生物组成 (包括突变体) 的操作、植物生长的监测、根系形态、渗出物组成和受控环境条件下的微生物定位。我们预计, 这些详细的协议将成为其他研究人员的一个重要的出发点, 最好的帮助建立标准化实验系统, 以调查植物微生物相互作用。

每个 EcoFAB 系统包括一个 EcoFAB 设备和一个工厂大小的透明塑料容器。一个 EcoFAB 设备有一个植物水库, 一个根生长室, 一个1.6 毫米流量入口, 1.6 毫米出口为标准 EcoFAB 设备 (图 2D &图 3H) 或一个10毫米出口为宽出口 EcoFAB 设备 (图 2F &图3I).该植物储层设计的梯形形状, 具有6毫米的顶部开口和 3 mm 底部开放, 这一设计减少了流动泄漏的机会, 在液体注入, 并确保足够的空间, 以促进植物生长。根生长室采用具有2毫米深度的椭圆形形状, 适合许多模型植物的根系统, 如图 2C和E所示。标准 EcoFAB 装置的入口和出口通道都可以与 PTFE 油管连接 , 因此养分溶液可以在不打开 EcoFAB 容器的情况入根生长室。宽出口 EcoFAB 装置大大降低了出口的流动阻力, 最好是在植物生长较粗的根系或周期性地收集根分泌物后, 在复杂的根系系统中产生的。
在设计软件中创建了用于制造 EcoFAB 设备的注塑模, 然后3D 打印在刚性不透明 photopolymers 中, 如图 2和图 3所示。EcoFABs 内的植物可以使用长的工作距离直接观察显微镜, 确保无菌生长环境 (图 8A,补充文件 1)。EcoFAB 设备也可以适应高分辨率的显微镜阶段, 使植物-微生物相互作用的更高分辨率成像 (图 8B,补充文件 2)。不育在这种环境下不保持, 高分辨率成像因此仅适用于端点测量。
EcoFABs 的设计是为了能够对植物进行系统的研究, 例如它们的形态、新陈代谢和微生物群落在其生命周期的不同生长阶段。在这里, EcoFABs 被视为研究各种植物种类的一般平台。图 8C-e显示7天的老拟南芥、Brachypodium distachyon和柳枝柳枝稷在 EcoFABs 中生长。三株植物在 EcoFAB 中生长良好, 超过一个月。双子叶、拟南芥和单子叶植物、 Brachypodium distachyon 均被发现在 EcoFABs 中达到繁殖阶段。
可逆密封系统允许在 EcoFABs (步骤 2.2) 内使用固体基底 (e. g)。这种可逆的密封方法能够将固体基底装入根系生长室, 并能从根豆科的特定区域中采集样本。图 8FH显示一组14天的老Brachypodium distachyon , 它们生长在水培培养基中, 还有沙子和土壤, 辅以水培培养基 (沙子) 和水分 (土壤)。根生长腔中的薄固体基底层允许光线穿透, 用于显微成像的根系统。
根形态学被定义为植物根系的空间构型和分布, 并已被批准为各种生长环境的必要生理反应, 如养分或水的可用性32,33, 34。EcoFABs 提供了一种方便的方法来研究植物形态学的时间或在不同的营养条件下。图 9 a-f显示了一个示例, 使用 EcoFABs 在头三周内跟踪Brachypodium distachyon的根形貌。一个Brachypodium distachyon苗被转移到 EcoFAB 装置中, 其根结构由一个摄像机在生物 RAD 凝胶成像仪中记录下来。图像处理程序 (如图像 J、python 和 matlab) 可以进一步应用于时间或不同介质环境下的根形貌变化的量化。在三周的过程中, 总根面积的量化显示在早期 (< 1 周) 的逐渐增加, 其次是线性增长趋势到三周结束, 如图 9G所示。
构建 EcoFAB 的主要动力是研究植物-微生物的相互作用。如步骤5.4 所述, 微生物通过入口通道转移到 EcoFAB 装置的根生长室。图 10显示了一个包含假单胞 simea (以前为、荧光) WCS417 (WCS417) 的 EcoFAB, 该植物生长促进细菌与化学发光标签, 被添加到植物根系系统中, 浓度为每株10个6个单元格。用凝胶成像仪检测 WCS417 信号, 表明根系生长室中 WCS417 微生物的空间分布明显。在两个 MS 液体介质与和没有沙子固体基体, WCS417 微生物殖民地整个根系统的表面与微生物集中在根尖端区域, 可能由于根尖的活跃营养素生产 (图10G & H)35。另一方面, 土壤基质中的 WCS417 微生物聚集在植物库区周围, 而不是根尖 (图 10I)。当微生物通过出口通道添加时, 微生物也能够在土壤基质中移动, 但在根部没有积聚, 如在有或没有沙子的液体介质中观察到的。这可能表明, 土壤是一个足够的养分来源, 和微生物迁移到植物水库的最佳呼吸条件。
为了研究植物根系分泌物的代谢产物以及植物-微生物相互作用的代谢物吸收和释放, 在 EcoFABs 植物的不同生长阶段收集了根系生长室的渗出液。如步骤6所述, 然后提取渗出物样本进行 LC-MS 分析。利用这种方法, 检测出了由植物散发出来的一系列代谢产物, 并对微生物的消耗进行了研究, 并对具有微生物定植和不带细菌的根分泌物进行了相关的代谢产物分析。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57170/57170fig1.jpg"> 图 1: EcoFAB 工作流.植物发芽在盘子上, 转移到灭菌的 EcoFAB, 微生物可以添加。无损抽样: 根分泌物可以取样和成像, 根形态学可以可视化。破坏性取样可以详细分析微生物、根和射击参数。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57170/57170fig2.jpg"> 图 2: 用于 EcoFAB 设备制造的3D 打印模具的组件.(a) 铸造框架的顶部和倾斜视图。(B) 插入物的顶部和倾斜视图。(C) 标准模具底座的顶部和倾斜视图。(E) 宽出口模具底座的顶部和倾斜视图。(D、F)用于制造标准和宽出口 EcoFAB 装置的装配模具。椭圆尺寸为51毫米 x 34 毫米为小 EcoFAB 模子和76毫米 x 62 毫米为大 EcoFAB 模子。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig2large.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/57170/57170fig3.jpg"> 图 3: EcoFAB 设备制造.(A) 将弹性体底座和固化剂的混合物倒入模具中。(B) 以85摄氏度的混合物将模具加热4小时 (C) 除去模具中的插入物。(D) 将该硅烷与铸造框架分离。(E) 将模具底座推出铸件框架。(F) 使用刀将该硅烷与模具沿边缘分离。(G) 在模具底座上慢慢剥离该基膜。(H) 为标准的两层的入口和出口通道戳孔。(I) 为宽出口的该层的进口通道戳一个孔。(J) 由15:1 个弹性体基础组成的树脂层 (用于固化剂混合物) 和显微镜滑道冲洗, 并转移到等离子清洗剂中进行粘合。(K) 使用夹具将所用的基片 (由5:1 弹性体底座制成的固化剂混合物) 固定在显微镜上。(L) 直接在显微镜上滑动 (由30:1 个弹性体底座制成, 以固化混合剂)。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig3large.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/57170/57170fig4.jpg"> 图 4: 自定义夹具的设计.(a) 顶部夹板的顶部和倾斜视图。(B) 底部夹板的顶部和倾斜视图。(C) 四套六角帽螺钉装配钳的顶部和倾斜视图。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57170/57170fig5.jpg"> 图 5: 安装 LED 增长灯.(a) 将9个 LED 剪辑的位置标记为 EcoFAB 容器周围的螺旋形。(B) 附在 EcoFAB 容器上的 LED 夹子。(C) 通过这些剪辑对 LED 带进行线程处理。(D) 将 LED 带连接到与24V 电源相连的控制器上。(E) 与控制器连线的示意图。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57170/57170fig6.jpg"> 图 6: 将幼苗转移到 EcoFABs.(a) Brachypodium distachyon植物在 0.5 MS 板上生长2天。(B) 用植物生长培养基填充根腔。(C) 使用镊子将根部小心地插入植物油藏。(D) 在容器底部添加3毫升水后, 用微孔胶带密封 EcoFAB 容器。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57170/57170fig7.jpg"> 图 7: EcoFABs 中植物根的随着 nims 成像。(a) 在无菌 EcoFAB 中生长的Brachypodium distachyon 。(B) 用铜带将随着 nims 芯片连接到自定义 MALDI 板上, 并将其加载到 MALDI 质谱仪中, 将随着 nims 芯片与该植物连接。(d G) 一个7天的旧的和一个20天的老Brachypodium distachyon植物用于随着 nims 成像 (d, E) 和相应的随着 nims 图像 (F, g)。主要离子以红色、绿色和蓝色突出显示。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57170/57170fig8.jpg"> 图 8: EcoFABs 的一般应用.(A) 在具有长工作距离显微镜设置的 EcoFAB 中直接捕获Brachypodium distachyon的根增长。(B) 通过高分辨率显微镜的设置直接观察根微生物的相互作用。(E) 7 天旧的拟南芥(c), Brachypodium distachyon (D) 和柳枝柳枝稷(E) 在 0.5 ms 水培培养基中, (f H) 14 天老Brachypodium distachyon在 0.5 ms 水培 (f) 中生长, 在沙子 (G) 和土壤 (H)基材分别提供 0.5 MS 介质和水。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57170/57170fig9.jpg"> 图 9: 使用 EcoFABs 研究根形态学.(F)Brachypodium distachyon的根发育在 EcoFABs 三周内填充 0.5 MS 培养基: (A) 2 天, (B) 4 天, (C) 7 天, (D) 11 天, (E) 14 天, (F) 21 天的增长。(G) ImageJ 软件估计平均根表面积。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/57170/57170fig10.jpg"> 图 10: 使用 EcoFABs 研究根微生物相互作用.(A、B、C)一组15天的旧Brachypodium distachyon以假单胞菌荧光WCS417 在不同形式的介质-MS 液体溶液、沙子和土壤基底上进行殖民化。(D、E、F)他们的根系统的明亮的领域图片。(G、H、I)在14天的共同栽培后, 这些根系的相应的化学发光图像。<a href="http://cloudflare.jove.com/files/ftp_upload/57170/57170fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助文件1。使用 EcoFAB 捕获根增长.<a href="http://cloudflare.jove.com/files/ftp_upload/57170/video1_capture_growing_roots_(2).mp4">请单击此处下载此文件.</a>
辅助文件2。使用 EcoFAB 捕获根微生物相互作用.<a href="http://cloudflare.jove.com/files/ftp_upload/57170/video2_capture_root_microbes_interaction.avi">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions at JoVE.com

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

本文描述了设备 (EcoFABs) 的生态系统制造的详细协议, 它能够在高控制的实验室条件下对植物和植物微生物相互作用进行研究。

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

ecosystem-fabrication-ecofab-protocols-for-construction-laboratory

Research

JoVE Journal

Environment

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

18.3K 次观看.  Lawrence Berkeley National Laboratory. 本文描述了设备 (EcoFABs) 的生态系统制造的详细协议, 它能够在高控制的实验室条件下对植物和植物微生物相互作用进行研究。

视频: Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

科研

环境科学

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

<meta charset="utf8"></head><body>模拟甲烷菌生态位 – 梯度注射器模型生态系统

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

<meta charset="utf8"></head><body>作者聚焦:设计简单且廉价的技术在实验室中培养甲烷氧化细菌

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

JenaTron - An Experimental Approach to Study the Effects of Plant History and Soil History on Grassland Ecosystem Functioning

The global loss of biodiversity has motivated many studies that experimentally vary plant species richness and examine the consequences for ecosystem functioning. Such experiments generally show a positive relationship between above- and below-ground biodiversity and the functioning of terrestrial ecosystems. Moreover, this relationship tends to strengthen over time, seen as enhanced functioning of diverse plant communities and reduced functioning of low-diversity plant communities. Differences in multitrophic community assembly and biotic interactions in high- versus low-diversity plant communities are hypothesized to affect plant performance by altering consumer community structure and function and driving plastic or micro-evolutionary responses of plant species in the plant communities. To resolve this complex interplay of community history, we separated these effects into plant and soil history. Plant history refers to all plant-level responses to past abiotic and biotic selection pressures experienced in their communities, while soil history relates to all abiotic and biotic soil properties developed as a legacy of plant-soil interactions under variable plant diversity. We set up a biodiversity experiment in an Ecotron, a terrestrial mesocosm facility that allows controlling environmental conditions above- and below-ground, to test whether the strengthening biodiversity-ecosystem functioning relationship is due to soil history, plant history, or a combination of both. We established a plant diversity gradient consisting of 1, 2, 3, and 6 grassland plant species and factorially nested with soil history and plant history treatments for each level of plant species richness. Representative results demonstrate the successful establishment of target treatments in the Ecotron experiment, observing the effects of plant and soil history on initial plant development and final plant growth. Additionally, we provide a case study for data analysis of individual response variables. We outline research objectives and methods to comprehensively assess the multifunctional responses to the experimental treatments necessary to ultimately address the overarching hypothesis.

Here we present a study established in the iDiv Ecotron, terrestrial mesocosm chambers with controlled environmental conditions above- and below-ground, to study the independent and interactive effects of plant history and soil history on ecosystem functioning of grassland communities along a plant diversity gradient.

JenaTron - 一种研究植物历史和土壤历史对草原生态系统功能影响的实验方法

The global loss of biodiversity has motivated many studies that experimentally vary plant species richness and examine the consequences for ecosystem ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

生物学

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

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

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 ...

生物工程

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

摘要

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此视频中的章节

用于操纵植物诱导土壤异质性的实验协议

多年生草试验中土壤、根际和根系微生物群落的分离与分析

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

JenaTron - 一种研究植物历史和土壤历史对草原生态系统功能影响的实验方法

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

微孔阵列平台中微生物群落发展的组装与跟踪

生成受控动态化学景观，研究微生物行为