这项工作得到了国家卫生研究院授予GM118085的支持，部分得到了罗伯特·韦尔奇基金会的支持（授予R-1811R.M.H.）。

1. 在缺乏活动力的菌株中分离抑制突变体
注:使用此方法作为广泛的"捕获所有"，以确定动能缺陷的一般性质。
<ol><li>软加板准备 注:软阿加，也被称为运动或游泳的阿加，是一个低百分比的阿加（+0.2-0.35%w/v），长期用于检测化疗31，32。<ol>
<li>在 2 L 圆形底烧瓶中加入 3 克 bacto-agar（0.3% w/v） 和 20 克 LB。在烧瓶中加入 1 L 的 ddH2O（双蒸馏水），然后使用搅拌棒和磁搅拌板均匀地混合悬架。</li>
		<li>高压灭菌 20 分钟，121 °C。</li>
		<li>使用上面的杆/板，让温和的搅拌冷却。当温度达到约 50 °C 时，将 25 mL 倒入无菌的 Petri 盘（100 mm x 15 mm），并允许熔融的 agar 将盖子固定至少 1 小时，用于 16 小时内。</li>
	</ol></li>
	<li>抑制突变体的文化准备、接种和隔离 注: 大肠杆菌 在营养丰富的介质中心接种，用软醋凝固，在当地消耗营养物质，从而产生它们遵循的营养梯度。当它们向外移动时，定义的"环"出现（图1A），这与细菌对特定的化学作用有关。化疗系统或旗杆菌电机结构部件的缺陷可能会损害此检测中的性能。通常，具有活动优势的突变体在筛选过程中出现，并且可以看到从单点或环外围的多个点出现，从它们"燃烧"出来的地方（图1C）。人们会注意到，游泳前部最外侧的边缘很容易与未殖民的处女软阿加形成对比。<ol>
<li>在 5 mL 的伦诺克斯兄弟 （LB; 10 g/L 试剂酮， 5 克/升酵母提取物， 和 5 克/升 NaCl， 材料表） 在 30 °C 水平摇晃 （220 r.p.m.第二天，亚文化（1:100稀释）在新鲜LB，在相同的条件下增长到指数阶段（OD600 的0.6）。</li>
		<li>使用移液器将 6 μL 的培养物接种到软醋板 （1.1） 的中心，将装载的无菌尖端推入 Agar 以轻轻排出其内容。转移到30°C并孵育（图1B），直到运动"火焰"明显，从接种点或运动环的外围发出，通常在24-36小时（图1C）。 注:在动感分析中，在突变隔离物旁边接种野生菌株进行比较。这种野生型菌株将显示出典型的同心化学环（图1A），并在8-10小时内填充板。</li>
		<li>使用无菌线环将细胞从"火焰"区域和条纹中提升，将单个菌落纯化到 LB 硬阿加板上（如上所示，用 15 克/升的 bacto-agar 凝固）。</li>
		<li>使用无菌线环从条纹板中挑选单个菌落，并通过单个菌落的条纹重新净化，以确保隔离"纯"菌落隔离。</li>
	</ol></li>
	<li>抑制突变体的确认和特征
	<ol>
<li>确认孤立的抑制突变体已恢复活力。准备软加板（1.1），以及感兴趣的菌株（如1.2.1），包括野生类型和开始的"缺乏活力"菌株进行比较。</li>
		<li>在 30 °C 下接种板（如 1.2.2），在 8-10 小时孵育。</li>
		<li>记录最外环（圆的边缘）的直径，并比较确定哪些隔离物已显著恢复活力。 注:建议在整个实验过程中拍摄板材。为了获得最佳效果，请使用"光桶"设备30，其中数码相机安装在光源上方，以便更好地照明，以测量游泳殖民地的直径，并将其与未殖民化的偶发体区分开来。</li>
		<li>经验证的受试者可根据需要与 WGS 隔离，从而获得足够的"序列覆盖"，从而积极识别恢复野生类型功能的突变。</li>
	</ol></li>
</ol>2. 通过细胞系绳量化旗杆菌运动行为
注意:当正常运行翻滚行为（化学轴）出现受损时，使用此方法。
<ol><li>
文化准备和旗杆切口
	<ol>
<li>准备第 1.2.1 步中描述的兴趣应变的指数阶段文化。</li>
		<li>以 2，000 x g 离心方式将 10 mL 细胞颗粒 10 mL，在 3 分钟内再悬浮在 10 mL 的过滤灭菌运动缓冲器 （MB） 中: 10 mM 磷酸钾缓冲器 [0.0935 M K2HPO4， 0.0065 M KH2PO4， pH 7.0]， 0.1 mM EDTA [pH 7.0]， 10 mM NaCl， 75 mM KCl）. 注:MB支持 活性， 但不支持细菌生长</li>
		<li>重复步骤 2.1.2 两次，然后在 1 mL 的 MB 中重新暂停最后颗粒。</li>
		<li>将电池悬架转移到 1 mL 注射器中，并将 23G 针头连接到末端。组装相同的注射器/针具，并通过6英寸的聚乙烯管（内径0.58毫米）将两者连接在一起，紧紧地套在每根针尖上。</li>
		<li>通过轻轻地将细胞悬架从一个注射器来回传递到其他 50 倍，每 10 次通过之间暂停 1 分钟，从而剪切旗杆（它们很脆弱，很容易断裂）。</li>
		<li>将切变电池以 2，000 x g 离心 3 分钟，最后卷为 500 μL MB。</li>
	</ol>
</li>
	<li>
幻灯片制备和细胞系绳
	<ol>
<li>通过将 18 mm x 18 mm 的盖片堆叠在 3 英寸 x 1 英寸 x 1 毫米玻璃显微镜滑动上，通过双面胶带（图 S1）分离，准备细胞固定室。</li>
		<li>通过应用到腔室顶部，用 0.01% （w/v） 多赖氨酸溶液冲洗腔室。将底部边缘（覆盖唇与显微镜滑动一起冲洗）倾斜到任务雨刷器（纸巾）上，以帮助通过腔室绘制溶液（图 S1）。然后在室温下孵育10分钟。</li>
		<li>使用第 2.2.2 步中描述的 40 μL MB 清洗腔室三次</li>
		<li>将 40 μL 的剪切细胞悬架（上图所示）添加到腔室顶部，并在室温下孵育 10 分钟，使细胞附着在盖唇上。</li>
		<li>轻轻冲洗腔室与 40 μL 的 MB 在 2.2.3 中删除未连接的细胞。</li>
	</ol>
</li>
	<li>
细胞旋转记录和量化
	<ol>
<li>将装有系绳细胞的显微镜滑梯转移到显微镜阶段。</li>
		<li>使用相对比显微镜和100倍目标，扫描人口，寻找固定到位的细胞，并在单轴上旋转，即在固定点上平稳旋转，而不是以细胞进出聚焦的角度呈现（视频1）。</li>
		<li>使用商用显微镜和相关摄像机。打开相关软件，确保感兴趣的单元格处于对焦状态，并单击 视频采集 以录制一分钟的单元格旋转（每秒 10 帧或更高）。</li>
		<li>从视频播放中，量化每分钟完整旋转次数和细胞改变方向（切换频率）的次数。 注:旋转速度和切换频率可能太快，无法用眼睛测量，因此建议使用视频软件提供慢/精细播放，或采用自动化软件系统来量化旋转模式33。另一种选择是使用甲基纤维素（或类似剂）增加MB的粘度，以帮助减缓和解决更快的细菌的旋转，或在使用帧速率低的相机时进行补偿。</li>
		<li>重复步骤 2.3.2 与生物复制，以编制感兴趣的人口的表示。</li>
	</ol>
</li>
</ol>3. 在过境分析中准备成群
注意:使用此方法评估突变或条件对群体活力的影响。沼泽-阿加是指通常高于软阿加的百分比的阿加。在软阿加（0.3%）中，细胞在亚加体内单独游泳。在群加（0.5%及以上）中，细胞在表面上作为一组移动。虽然蜂群板必须在这里详细使用，但游泳板的保质期更长，并且可能使用数天。我们的个人喜好是在1-2天内使用。
<ol><li>
准备群阿加
	<ol>
<li>在 2 L 圆形底烧瓶中加入 5 克 Eiken agar（0.5% w/v） 和 20 克 LB。在烧瓶中加入 1 L 的 ddH2O，然后使用搅拌棒和磁搅拌板均匀地混合悬架。</li>
		<li>高压灭菌 20 分钟，121 °C。</li>
		<li>允许用温和的搅拌冷却，以避免使用上面的杆/板的任何气泡。当大约 50 °C 时，添加过滤消毒葡萄糖，最终浓度为 0.5%。</li>
		<li>将 25 mL 倒入无菌的 Petri 菜肴（100 mm x 15 mm），并允许在室温下设置至少 14 小时且不超过 20 小时。不要存储供将来使用。</li>
	</ol>
</li>
	<li>
蜂盘的接种和孵化
	<ol>
<li>通过在 agar 顶部发现 6 μL 的中指数文化（如 1.2 中准备）接种。</li>
		<li>将盖子关闭 5-10 分钟，当接种管干燥到阿加表面时更换。</li>
		<li>在 30 °C 下孵育 8 小时。通过移除盖子来避免检查群进度的诱惑，因为这将有助于干燥的阿加和受损的蜂群。 注:孵化时间可能因应变表型而异。一些孤立的突变可能会阻碍成群的能力，需要降低加糖百分比或更长的潜伏期。</li>
	</ol>
</li>
	<li>
准备过境检测板 注:此检测使用改良的培养皿，其中塑料隔（边）创建两个腔室，而不是一个腔室（图 2A）。每个腔室可以独立于其他房间准备，在"连接"两个房间之前，为蜂群提供不同的条件。根据实验设计，第一个腔室（指定左）可以准备与游泳的阿加（0.3%w/v）或群阿加（0.5%w/v），从那里细菌可以越过边界迁移到右室含有蜂群阿加+/-任何必要的补充或挑战（例如，抗生素）。对任何一种阿加的迁移通常通过记录细菌殖民化（边缘到边缘）从原始接种点的最宽直径来测量/比较。<ol>
<li>如果需要，准备 1.1 中描述的游泳阿加。</li>
		<li>准备3.1中描述的群加。</li>
		<li>将 +30 mL 的蜂群阿加（如果需要，需要补充）倒入双隔间 Petri 盘（100 mm x 15 mm）的右室，使其与腔室之间的塑料分隔器平起平落，但不会溢出到左侧（图 2B）。</li>
		<li>在阿加变硬后，用+30ml的游泳或蜂群的阿加填充左室，再次到与塑料隔线接触点（图2C）。在设置之前，使用无菌移液器尖端轻轻地将阿加拖过边界，将两侧连接起来，连接横跨整个鸿沟长度的 +1 毫米高的阿加桥（图 2D）。</li>
		<li>让盘子在室温下干燥（3.1）。 注:创建桥梁的另一种方法是让左室醋干燥，然后沿着塑料分隔器缓慢移液器 +100 μL 熔融群醋，以连接两个腔室 （3.4）。在 30 °C 孵育 12-16 小时之前，或直到蜂群在右腔上取得足够进展，以便比较感兴趣的菌株之前，如上文所述，为左室的盘子接种疫苗（1.2.2）或群（3.2）agar。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

抑制剂突变的分离和定性成功地帮助识别了化疗系统35、36、37以及电机机械本身38、39、40的关键部件。在使用第1号议定书时，必须包括多个独立复制，以确保隔离大量可能的突变，以弥补活动性损失。通过将培养体划入一条线而不是一个点来增加细菌的数?...

细菌在其生态利基中利用许多附属物进行运动和分散。弗拉盖拉驱动的动能是其中最快的，促进有利地区响应环境信号的殖民化，并大大有助于一些物种2，3的致病能力。旗菌可以单独在散装液体中游泳，或集体聚集在半固体表面4。细胞外旗手附着在膜内，由嵌入膜的旋转电机驱动，该电机利用离子梯度的功率产生扭矩，导致旋转 1、2、4、5、6、7、8。在大肠杆菌中，电机以恒定的扭矩9运行，电机输出可以按旋转速度和转子在逆时针 （CCW） 和顺时针 （CW） 方向之间的切换进行分类。CCW 旋转促进形成一个连贯的旗杆束，推动细胞向前（运行），而旋转方向 （CW） 的瞬时开关导致束部分或完全拆解10，细胞重新定位其游泳方向（翻滚）。大肠杆菌通常运行一秒钟，跌落十分之一秒。转子或"翻滚偏置"的切换频率由化疗信号系统控制，其中转子切除器检测外部化学信号并通过磷化物将其传输到旗杆电机，以延长运行以响应吸引物，或抑制它们以响应有毒化学物质11，12。游泳运动在0.3%的软性啤酒中测定。
在蜂拥而至时，细菌在半固体表面作为密集的集体导航，细菌群在连续旋转运动中流淌2、13、14、15。大肠杆菌群表现出化学感官生理变化（较低的翻滚偏差），更高的速度，和更高的耐受性抗菌细胞在散装液体中游动16，17。沼泽人部署的策略，以帮助运动，包括表面活性剂生产，恶性通货膨胀，和细胞拉长2。沼泽为细菌提供了在生态和临床环境中的竞争优势18，19，20。有两类蜂群细菌:温带蜂群，它只能聚集在媒体凝固与0.5-0.8%的阿加，和强大的蜂群，它可以导航通过更高的阿加浓度21。
存在各种分析来询问游泳的活力及其规定。当受到突变或环境条件的损害时，活动本身为识别功能增益抑制剂突变提供了强有力的选择。这些抑制剂可以是原始突变的真正还原剂，也可以是伪反变剂，其中第二个突变会恢复功能。这种突变体可以通过全基因组测序（WGS）来识别。不偏不倚的抑制器选择的替代方案是偏颇的靶向突变策略（例如 PCR 突变）。这些方法往往揭示了动能装置的功能或环境调节。如果目标是研究运动功能，那么用软糖测量的野生型运动性的恢复不一定意味着野生型运动输出的恢复。细胞系绳检测，其中细胞通过单个旗杆连接到玻璃表面，随后对细胞体的旋转进行监测，可以作为评估运动行为的初始选择。虽然现在可以采用更复杂的方法来监控电机性能，但所需的高速摄像头设置和用于运动分析的软件包限制了其广泛使用 22、23、24、25。细胞系绳检测仅要求剪切旗杆，允许将短丝附着在玻璃滑梯上，然后录下细胞体的旋转。虽然记录的电机速度在本次测定中较低，因为细胞体对旗手施加了高负荷，但此测定还是有助于对化学反应 26、27、28、29 进行有价值的见解，并且仍然是下面讨论的有效调查工具。
沼泽的活力给研究人员带来了一系列不同的挑战。功能抑制器的选择只在产生大量表面活性剂的群中有效，并且容易地蜂拥而至。表面活性剂非生产者，如大肠杆菌是挑剔的选择，在阿加的选择，媒体组成和湿度的环境2，13，14，21。一旦蜂群条件建立，过境分析17是一个有用的方法来询问群导航新的/苛刻条件的能力。虽然下面提出的协议与大肠杆菌有关，但它们很容易适应其他物种的应用。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Dehydrated Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-15-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt, Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>02-002-786</td>
			<td></td>
		</tr>
		<tr>
			<td>Eiken agar</td>
			<td>Eiken Chemical Co. Japan</td>
			<td>E-MJ00</td>
			<td>Essential for E. coli swarming</td>
		</tr>
		<tr>
			<td>Glucose D (+)</td>
			<td>Fisher Scientific</td>
			<td>410955000</td>
			<td></td>
		</tr>
		<tr>
			<td>LB (Lennox) Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1427-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution (0.1%)</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>18-605-496</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate dibasic (K2HPO4)</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellSense microscope imaging software (V. 1.6)</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent software for microscope used</td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Scotch 666 Doube Sided Tape</td>
			<td>Fisher</td>
			<td>50-285-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted microscope slides 3x1x1mm</td>
			<td>Fisher</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX53 microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td>Any upright or inverted phase microscope can be used</td>
		</tr>
		<tr>
			<td>Petri dishes (100 mm diameter)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>For soft-agar assays</td>
		</tr>
		<tr>
			<td>Polyethylene Nebulizer Capillary Tubing (0.58mm x 99mm 3.0m)</td>
			<td>Perkin Elmer</td>
			<td>9908265</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Petri Dish with 2 Compartments</td>
			<td>VWR</td>
			<td>89200-944</td>
			<td>For border-crossing assays</td>
		</tr>
		<tr>
			<td>Safety Hypodermic Needles (23G)</td>
			<td>Fisher Scientific</td>
			<td>14-826A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe - 1 mL</td>
			<td>Fisher scientific</td>
			<td>14-955-450</td>
			<td></td>
		</tr>
		<tr>
			<td>Task/Tissue wipes</td>
			<td>Fisher scientific</td>
			<td>06-666</td>
			<td>Or equivalent single use tissue wipes</td>
		</tr>
		<tr>
			<td>VWR micro cover-glass 18x18mm</td>
			<td>VWR</td>
			<td>48366205</td>
			<td></td>
		</tr>
		<tr>
			<td>XM10 camera</td>
			<td>Olympus</td>
			<td>XM10</td>
			<td>Or equivalent microscope camera</td>
		</tr>
	</tbody>
</table>

investigating flagella-driven motility in escherichia coli by applying three established techniques in a series

活力对许多细菌物种的生存和成功至关重要。存在许多方法来利用动感来理解信号通路，阐明旗手部件的功能和组装，并检查和理解运动模式。在这里，我们演示了其中三种方法的组合。软性阿加的活力是最古老的，为分离体弱化菌株中的功能增益抑制剂突变提供了强有力的选择，通过第二次突变恢复活力。细胞系绳技术首先用于演示旗手电机的旋转性质，可用于评估信号效应器对电机速度的影响及其切换旋转方向的能力。"越境"检测是最新的，游泳细菌可以准备过渡到集体移动成群。综合起来，这些协议代表了一种系统而有力的方法，用于识别运动机械的部件，并描述它们在游泳和蜂拥而至的不同方面的作用。它们可以很容易地适应研究其他细菌物种的动能。

我们实验室34的最新研究详细介绍了大肠杆菌株中伪还原体的分离，其活动性因信号分子c-di-GMP的高水平而受损。这种菌株（JP1442）有两种突变:yhjH和+ycgR。YhjH是大肠杆菌中降解c-di-GMP的最活性磷二酯酶。缺乏 Yhjh 会导致 c - di - gmp 水平升高和抑制动能。Ycgr 是 c - di - gmp 效应器。在与 c-di-GMP 的复合体中，YcgR 与旗杆转子结合，首先诱导 CCW 电机旋转，然后...

Watch this Scientific Journal Video about Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series at JoVE.com

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

许多细菌使用旗菌驱动的活力来导航他们的环境，并单独和集体殖民有利的环境。这里展示的是使用三种既定的方法，利用动感作为选择工具，以确定有助于游泳和蜂拥而至的活力的组件/路径。

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

investigating-flagella-driven-motility-escherichia-coli-applying

Research

JoVE Journal

Biology

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

7.4K 次观看.  The University of Texas at Austin. 许多细菌使用旗菌驱动的活力来导航他们的环境，并单独和集体殖民有利的环境。这里展示的是使用三种既定的方法，利用动感作为选择工具，以确定有助于游泳和蜂拥而至的活力的组件/路径。

视频: Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

科研

生物学

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157:H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性