这项工作得到了美国国立卫生研究院资助R35GM128873和美国国家科学基金会资助2045493（授予MNM）的支持。梅根·妮可·麦克莱恩（Megan Nicole McClean）博士在巴勒斯惠康基金会（Burroughs Wellcome Fund）的科学界面（Scientific Interface）上获得了职业奖。Z.P.H. 得到了 NHGRI 对基因组科学培训计划 5T32HG002760 的培训资助。我们感谢与麦克莱恩实验室成员进行的富有成效的讨论，特别是感谢基兰·斯威尼（Kieran Sweeney）对手稿的评论。

本研究中使用的酵母菌株记录在 材料表中。这些菌株在 22 °C 至 30 °C 的温度范围内表现出强劲的生长，并且可以在各种标准酵母培养基中培养。
1. 设置自动化工作站
<ol><li>为自动化工作站配备能够移动微孔板的机器人夹持臂（RGA，参见 材料表）（图 1）。</li>
	<li>将微孔板加热器振荡器（见材料表）安装到自动化工作站（图1（1））中，并带有提供RGA访问的自动板锁定机构。</li>
	<li>将酶标仪放置在自动化工作站附近（图 1（2）），确保 RGA 可访问性。</li>
	<li>结合微孔板照明装置（图1（3）），方便RGA访问。示例包括 optoPlate8 （如本研究中使用的）或 LITOS9 （参见 材料表）。</li>
</ol>2、照明装置的准备
<ol><li>按照既定方法8、10、11 构建和校准 optoPlate（或替代照明设备）。</li>
	<li>利用照明设备上的适配器启用 RGA 访问。</li>
	<li>使用电子表格输入10 或图形界面12 对 optoPlate 进行编程。按照步骤 3 执行光刺激程序。</li>
</ol>3. 设计光刺激程序
<ol><li>确定实验所需的光照条件。</li>
	<li>在电子表格中输入所需的光照条件，包括光强度、光开始时间、脉冲长度、脉冲数和脉冲间持续时间。 注意:使用 GitHub 存储库中提供的说明将此信息刷入 optoPlate:github.com/mccleanlab/Optoplate-96。请记住，样品板在酶标仪或加热器振荡器上不会被照亮。这些事件的持续时间和频率可能需要根据特定的实验要求进行优化。</li>
	<li>包括每个菌株的黑暗条件，以便进行适当的背景测量。</li>
	<li>对于评估转化体功能的初始表征实验，请使用高光强度。 注意:应针对更敏感的实验优化光强度，因为过多的光可能对酵母13 产生光毒性。</li>
</ol>4.酶标仪的准备
<ol><li>在进行实验之前，配置酶标仪以测量所需的目标量。 注:在本例中，酶标仪被配置为测量由目标菌株表达的报告基因的荧光。根据实验要求，可以使用其他输出，例如发光或光密度。</li>
	<li>在合成完全（SC）培养基14 （或另一种低荧光培养基）（参见 材料表）中培养目标菌株（连同非荧光对照）至要测量的最高细胞密度。将培养物转移到玻璃底黑壁微孔板中（参见 材料表）并测量它们以确定最佳的酶标仪设置。 注意: 通过正确输入板尺寸并从下方测量板来确保读数准确。</li>
	<li>参考荧光蛋白数据库（例如，fpbase.org）以获得目标荧光蛋白15的近似吸收和发射光谱。</li>
	<li>通过对含有荧光和非荧光菌株的孔进行z扫描来确定z值（板和读数器之间的距离）。选择产生最高信噪比的 z 值。</li>
	<li>通过对荧光和非荧光菌株进行吸收和发射扫描来优化吸收和发射光谱，以确定最佳信噪比。</li>
	<li>在增益设置为最佳水平的情况下测量荧光应变，确定可以使用的最高光学增益，而不会导致溢出测量误差。 注意:在涉及相同应变的实验中，应手动一致地设置此光学增益，以确保结果的一致性。</li>
	<li>在酶标仪软件中准备测量脚本（参见 图2）。该脚本将仪器配置为测量培养物的光密度和任何待测荧光蛋白的荧光光谱。 注:在600nm处测量菌株的光密度，除非它们表达红色荧光蛋白。对于表达红色荧光蛋白的菌株，测量 700 nm 处的光密度以避免偏差16。</li>
	<li>设置测量脚本以在测量过程中保持 30 °C 的内部孵育温度。</li>
	<li>根据偏好配置测量脚本以将数据导出到电子表格或 ASCII 文件中。</li>
</ol>5. 机器人编程
<ol><li>根据载体（例如，加热器振荡器、巢平台、照明设备）和实验室器皿（即 96 孔板）的物理布局，在自动化工作站软件中配置工作台定义（参见 材料表）。按照以下步骤在自动化工作站软件中创建一个脚本来执行光感应和测量（图 3）。</li>
	<li>禁用任何内部照明源，以防止光遗传学系统的背景激活。</li>
	<li>将加热器摇床设置为保持 30 °C 的温度。 当板不在加热器振动器上时，它将处于环境温度 （22 °C）。</li>
	<li>利用循环、计时器和循环计数变量定期重复诱导和测量细胞的步骤。</li>
	<li>在记录测量值之前，摇晃样品板以确保所有细胞的正确悬浮。以 1,000 rpm 的速度以 2 mm 的轨道振荡 60 秒通常足以重悬酿酒 酵母 细胞并避免测量偏差。</li>
	<li>使用机械臂将样品板移动到酶标仪上，并取下盖子（如果适用），以防止光密度测量出现偏差。如果酶标仪托架定义设置为不允许盖子，控制软件将自动将盖子取下并更换到指定位置。</li>
	<li>按照步骤 4 中的说明执行酶标仪测量脚本。</li>
	<li>盖上样品板上的盖子，然后使用机械臂将板移动到照明装置上。</li>
	<li>将脚本设置为等待，直到计时器达到指定的时间间隔（例如，30 分钟乘以循环计数变量），然后重复整个循环以获得所需的迭代次数（例如，48 小时实验为 24 次）。</li>
	<li>排除潜在错误，确保载体和实验室器皿定义的正常运行，以及 RGA 通过多次运行空板循环来准确拾取和放置板的能力。</li>
	<li>配置用户警报，以通知用户在协议执行期间可能发生的任何仪器状态更改或错误。</li>
</ol>6. 设置样品板
<ol><li>在富培养基平板上培养酵母菌株，例如YPD琼脂17 （参见 材料表）。包括非荧光（阴性）对照。</li>
	<li>从平板中选择菌落，并将它们接种到玻璃培养管中的 3 mL SC 培养基14 （或其他低荧光培养基，如 LFM18）中。将培养物在30°C下在滚筒上孵育过夜，同时将它们保持在黑暗或无响应的光条件下（例如，红光用于蓝光响应系统）。</li>
	<li>通过将每种培养物的200μL稀释到1mL的SC培养基中，并使用分光光度计或酶标仪记录600nm处的光密度（OD 600）来测量过夜培养物的光密度。</li>
	<li>在玻璃培养管中将每个过夜培养物稀释至OD600 0.1。如需更高通量的菌株测试，请在微孔板中进行自动稀释。</li>
	<li>将稀释的培养物移入 96 孔板中。为每种条件（即，具有相同应变和光照条件的三个相同孔）包括一式三份孔，以考虑技术差异。包括含有空白培养基和非荧光细胞的孔作为阴性对照，以测量背景荧光和光密度。</li>
	<li>在开始光诱导实验之前，将板在30°C下振荡孵育5小时。根据需要调整稀释量和孵育时间，以针对特定菌株和实验条件进行优化。</li>
</ol>7. 执行实验
<ol><li>将样品板放在加热器振荡器上，并按照步骤 5 中的说明启动自动化脚本。</li>
	<li>记录读板器上的初始测量值后，按照步骤 3 中的说明开始光刺激程序。</li>
	<li>如果自动化工作站不在黑暗的房间内，请用遮光窗帘遮住它，以避免背景照明。</li>
</ol>8. 数据分析
<ol><li>为实验创建电子表格图，反映 96 孔板的 8 x 12 布局。确保地图包括一个网格中要测量的应变的名称以及另一个网格中使用的光照条件的描述。</li>
	<li>利用 Python 脚本或其他首选编程语言来分析导出的数据。将地图电子表格导入到数组中，将菌株名称和条件名称与板的每个孔相关联。 注意:用于分析的示例代码可在以下位置找到:https://github.com/mccleanlab/Lustro。或者，可以使用应用程序来解析来自各种酶标仪19的数据。</li>
	<li>将导出的实验电子表格中的数据读入另一个数组。</li>
	<li>生成每种菌株和条件随时间变化的光密度值和荧光值图， 如图 4 所示。</li>
	<li>检查光密度图以确定培养物中指数增长或饱和的阶段。这些信息将有助于选择适当的时间点来比较菌株或条件之间的荧光测量， 如图5所示。</li>
</ol>

使用 Lustro 自动化技术进行酵母光遗传学反应分析

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作者没有什么可透露的。

本文介绍的 Lustro 协议可自动执行培养、照明和测量过程，从而实现光遗传学系统的高通量筛选和表征6。这是通过将照明设备、酶标仪和摇动设备集成到自动化工作站中来实现的。该协议特别证明了Lustro在筛选整合到 酵母酿酒酵 母中的不同光遗传学结构和比较光诱导程序的效用。
该协议中强调的几个关键步骤对于有效利用 Lustro 至关重要。仔细设计与正在研究的光遗传学结构的动力学相一致的定制光程序是必要的。此外，酶标仪的精确校准对于获得可靠的测量结果至关重要。在机器人上彻底进行实验，包括必要的调整，以确保与灯光程序的正确同步，对于确保脚本顺利运行至关重要。
此处提供的示例方案描述了在各种光刺激条件下驱动荧光报告基因表达的光诱导分裂转录因子与非荧光对照的比较。以30分钟的间隔从板中的每个孔中进行荧光测量，然后在测量前在加热器振荡器上振荡1分钟。如该协议所示，Lustro适用于集成到非贴壁细胞类型（包括细菌和其他酵母菌）中的蓝光响应光遗传学系统。然而，通过微小的修改，该协议可以很容易地扩展到不同的细胞类型、光遗传学系统和实验设计。对酶标仪设置的调整将允许测量荧光以外的输出，例如生物发光。对于需要更精细时间分辨率的应用，可以更频繁地进行测量。当对于需要振荡和温度控制的特定细胞类型至关重要时，可以更频繁地在加热器振荡器上重复孵育。结合气体和环境控制，例如通过孵化酒店，将能够包括哺乳动物细胞系。虽然这里描述的 Lustro 迭代使用特定的仪器，但 Lustro 平台可以很容易地适应其他实验室自动化机器人或酶标仪。LPA20 或 LITOS9 等照明设备可以替代 optoPlate 来刺激不同的光遗传学系统。Lustro 平台的未来修改可能涉及加入液体处理，以促进连续培养应用的自动稀释。这也将使 Lustro 能够适应网络遗传反馈控制，其中实时测量告知光或培养条件的变化，以实现或维持所需的响应 5,21,22。
高通量技术对于优化和利用光遗传学系统的动态特性至关重要。Lustro 克服了现有协议的许多局限性。例如，虽然基于生物反应器的光遗传学技术能够实现恒定的读数和培养条件，但它们的通量较低23,24,25,26。optoPlateReader27 设备有望在微孔板中进行实时光遗传学实验，但由于需要大量重复才能获得可靠的结果，因此目前通量较低，并且无法提供连续培养。另一方面，Lustro 能够对光遗传学系统进行高通量筛选，以表征其动态活性。尽管如此，Lustro 协议还是有一些局限性。Lustro 中的间歇性摇晃会导致酵母细胞6 出现小的生长滞后，但这可以通过调整照明设备以结合摇晃来解决。Lustro系统的另一个局限性是样品板在照明设备上不孵育，而是保持在环境温度（22°C）下。尽管每个样品的小体积允许高通量筛选，但当扩展到更大的反应体积用于生物生产或其他应用时，可能需要对照明步骤进行额外优化28,29。
总体而言，Lustro通过高通量筛选和精确的光控制促进了光遗传学系统的快速开发和测试。这种自动化方法能够在各种诱导条件下对不同的光遗传学结构进行有效的表征和比较，从而加快这些系统的迭代和改进。凭借其对不同细胞类型、光遗传学工具和自动化设置的适应性，Lustro 为光遗传学领域的进步铺平了道路，促进了动态基因表达控制的探索，并扩大了研究生物网络和工程细胞行为的可能性。

光遗传学是一种强大的技术，它利用光敏蛋白来高精度地控制细胞的行为 1,2,3。然而，光遗传学结构的原型设计和确定最佳照明条件可能非常耗时，因此很难优化光遗传学系统4,5。快速筛选和表征光遗传学系统活性的高通量方法可以加快设计-构建-测试周期，以对结构进行原型设计和探索其功能。
Lustro 平台是一种实验室自动化技术，专为光遗传学系统的高通量筛选和表征而设计。它将酶标仪、照明装置和振荡装置与自动化工作站6集成在一起。Lustro 结合了微孔板中细胞的自动培养和光刺激（图 1 和补充图 1），能够快速筛选和比较不同的光遗传学系统。Lustro 平台具有很强的适应性，可以推广到与其他实验室自动化机器人、照明设备、酶标仪、细胞类型和光遗传学系统（包括对不同波长的光有响应的机器人）配合使用。
该协议演示了Lustro用于表征光遗传学系统的设置和使用。以酵母中分裂转录因子的光遗传学控制为例系统，通过探测光输入与荧光报告基因 mScarlet-I7 表达之间的关系来说明该平台的功能和效用。通过遵循该协议，研究人员可以简化光遗传学系统的优化，并加速发现生物系统动态控制的新策略。

<table><tbody><tr><td>96-well glass bottom plate with&amp;nbsp; #1.5 cover glass</td><td>Cellvis</td><td>P96-1.5H-N</td><td></td></tr><tr><td>BioShake 3000-T elm (heater shaker)</td><td>QINSTRUMENTS</td><td></td><td></td></tr><tr><td>Fluent Automation Workstation</td><td>Tecan</td><td></td><td></td></tr><tr><td>LITOS (alternative illumination device)</td><td>Hohener, et al. Scientific Reports. 2022</td><td></td><td></td></tr><tr><td>optoPlate-96 (illumination device)</td><td>Bugaj, et al. Nature Protocols. 2019</td><td></td><td></td></tr><tr><td>Robotic Gripper Arm</td><td>Tecan</td><td></td><td></td></tr><tr><td>Spark (plate reader)</td><td>Tecan</td><td></td><td></td></tr><tr><td>Synthetic Complete media</td><td>SigmaAldrich</td><td>Y1250</td><td></td></tr><tr><td>Tecan Connect (user alert app)</td><td>Tecan</td><td></td><td></td></tr><tr><td>yMM1734 (BY4741 Mat&amp;alpha; ura3&amp;Delta;0::5&#039; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagB-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#039; homology&amp;nbsp; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td><td>Harmer, et al. ACS Syn Bio. 2023</td><td></td><td></td></tr><tr><td>yMM1763 (BY4741 Mat&amp;alpha; ura3&amp;Delta;0::5&#039; Ura3 homology, pRPL18B-Gal4DBD-CRY2(535)-tENO1, pRPL18B-Gal4AD-CIB1-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#039; homology&amp;nbsp; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td><td>Harmer, et al. ACS Syn Bio. 2023</td><td></td><td></td></tr><tr><td>yMM1765 (BY4741 Mat&amp;alpha; ura3&amp;Delta;0::5&#039; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagBM-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#039; homology&amp;nbsp; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td><td>Harmer, et al. ACS Syn Bio. 2023</td><td></td><td></td></tr><tr><td>YPD Agar</td><td>SigmaAldrich</td><td>Y1500</td><td></td></tr></tbody></table>

high-throughput optogenetics experiments in yeast using the automated platform lustro

光遗传学通过利用基因编码的光敏蛋白提供对细胞行为的精确控制。然而，优化这些系统以实现所需的功能通常需要多个设计-构建-测试周期，这可能既耗时又费力。为了应对这一挑战，我们开发了 Lustro，这是一个将光刺激与实验室自动化相结合的平台，能够对光遗传学系统进行高效的高通量筛选和表征。
Lustro 使用配备照明装置、摇晃装置和读板器的自动化工作站。通过使用机械臂，Lustro 使微孔板在这些设备之间自动移动，从而可以刺激光遗传学菌株并测量其反应。该协议提供了使用Lustro表征用于萌 芽酵母酿酒酵母中基因表达控制的光遗传学系统的分步指南。该协议涵盖了 Lustro 组件的设置，包括照明设备与自动化工作站的集成。它还提供了对照明设备、读板机和机器人进行编程的详细说明，确保在整个实验过程中顺利运行和数据采集。

图4A 显示了表达荧光报告基因的光遗传学菌株随时间变化的荧光值，该荧光报告基因由光诱导分裂转录因子控制。实验中使用的不同光照条件通过占空比的变化来反映，占空比表示灯亮起的时间百分比。观察到总荧光水平与光刺激的占空比成正比。 图4B 显示了同一实验的相应OD700 值。不同光照条件下光密度读数的一致性表明，该实验技术不会显着影响不同光照条件下菌株的生长速率。
通过测量荧光和光密度随时间的变化，与仅在单个时间点捕获输出的技术相比，可以更深入地了解光遗传学系统如何响应不同的光刺激程序。该时程数据对于选择特定时间点以比较不同的菌株和条件很有价值。 图5 显示了由不同光刺激程序诱导的两种不同光遗传学菌株的单个时间点（在光诱导10小时处测量）。两种菌株都采用光诱导的分裂转录因子来驱动荧光报告基因的表达。光脉冲强度、周期和占空比的变化在这些应变中会引起不同的反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65686/65686fig01.jpg"> 图 1:工作台布局和实验工作流程。 示例工作台布局的屏幕截图，表示 Lustro 中样品板的移动。板由机械臂从加热器振荡器（1）移动到酶标仪（2），然后移动到照明装置（3）。 照片见附图1。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/65686fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65686/65686fig02.jpg"> 图 2:读板器测量脚本。 将酶标仪设置为在30°C孵育并记录荧光和光密度测量值的读板器脚本的示例屏幕截图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/65686fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65686/65686fig03.jpg"> 图 3:自动化工作站脚本。 Lustro 的自动化工作站脚本的示例屏幕截图。该脚本启动一个计时器，确保关闭内部灯，将循环计数变量设置为初始值 0，并将加热器振荡器设置为在 30 °C 下孵育。 在每个循环中，将板锁定，摇动 1 分钟，移动到读板器，测量，然后移动到照明装置，并将机器人设置为等待 30 分钟循环间隔的剩余时间。在此时间结束时，循环计数器变量增加 1，并重复循环。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/65686fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65686/65686fig04.jpg"> 图 4:感应时间过程。 来自 Gal4BD-eMagA/eMagB-Gal4AD 分裂转录因子菌株和 pGAL1-mScarlet-I 报告基因 （yMM17346） 的光诱导时程数据样本。mScarlet-I7 的荧光在 563 nm 激发和 606 nm 发射下测量，光学增益为 130。光强度为 125 μW/cm2 ，误差条表示一式三份样品的标准误差。垂直红色虚线表示培养物何时达到饱和。（A） 应变随时间变化的荧光值。在所示实验的整个过程中重复光模式（如图所示）。插图显示，在整个调查过程中，轻脉冲时间穿插着暗脉冲时间。（B） （A） 中所示实验的光密度（在 700 nm 处测量）值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/65686fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65686/65686fig05.jpg"> 图5:不同光遗传学系统的比较。 CRY2（535）/CIB1 和 eMagA/eMagBM 分裂转录因子菌株与 pGAL1-mScarlet-I 报告基因（分别为 yMM1763 和 yMM17656）不同光诱导程序的比较。mScarlet-I7 的荧光在 563 nm 激发和 606 nm 发射下测量，光学增益为 130。除非另有说明，否则使用的光强度为 125 μW/cm2。误差线表示一式三份样本的标准误差（用点表示）。在诱导10小时后记录显示的荧光值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/65686fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图 1:Lustro 中使用的设备的代表性图像。 Lustro 设置的图片和所用设备的放大图像。在整个实验过程中，机械臂将样品板从加热振荡器移动到酶标仪，然后移动到照明装置。组件的编号在侧面带有图例。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65686/Harmer_2023_JoVE_Supplemental_Figure1.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Advancing Optogenetics Research Using Lustro at JoVE.com

作者聚焦:使用 Lustro 推进光遗传学研究 

该协议概述了利用自动化平台Lustro对酵母中的光遗传学系统进行高通量表征的步骤。

使用自动化平台 Lustro 在酵母中进行高通量光遗传学实验

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

目前有不同的设备，如生物反应器和微孔板照明器。生物反应器提供良好的时间分辨率，但通量有限。微孔板设备提供更高的通量，但缺乏自动化，无法随时间推移可靠地记录输出。Lustro 为光遗传学系统提供可靠和高通量的实时表征。它还可以与液处理机器人可以执行的检测连接。光遗传学系统是一种有吸引力的工具，因为它们对细胞行为提供了精细的空间和时间控制。更快地设计、构建和测试光遗传学系统的能力将为更好地理解和控制生物行为铺平道路。

high-throughput-optogenetics-experiments-yeast-using-automated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

605 次观看.  University of Wisconsin-Madison. 目前有不同的设备，如生物反应器和微孔板照明器。生物反应器提供良好的时间分辨率，但通量有限。微孔板设备提供更高的通量，但缺乏自动化，无法随时间推移可靠地记录输出。Lustro 为光遗传学系统提供可靠和高通量的实时表征。它还可以与液处理机器人可以执行的检测连接。光遗传学系统是一种有吸引力的工具，因为它们对细胞行为提供了精细的空间和时间控制...

视频: 作者聚焦:使用 Lustro 推进光遗传学研究 

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

遗传学

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

生物工程

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...