使用自动化平台 Lustro 在酵母中进行高通量光遗传学实验
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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）。 将微孔板加热器振荡器（见材料表）…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 96-well glass bottom plate with #1.5 cover glass | Cellvis | P96-1.5H-N | |
| BioShake 3000-T elm (heater shaker) | QINSTRUMENTS | ||
| Fluent Automation Workstation | Tecan | ||
| LITOS (alternative illumination device) | Hohener, et al. Scientific Reports. 2022 | ||
| optoPlate-96 (illumination device) | Bugaj, et al. Nature Protocols. 2019 | ||
| Robotic Gripper Arm | Tecan | Standard or long Z axes; regular gripper head or automatic Finger Exchange System gripper head, both with a choice of gripper fingers – eccentric, long eccentric, centric, tube; barcode reader option | |
| Spark (plate reader) | Tecan | ||
| Synthetic Complete media | SigmaAldrich | Y1250 | |
| Tecan Connect (user alert app) | Tecan | ||
| yMM1734 (BY4741 Matα ura3Δ0::5' Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagB-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3' homology his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5) | Harmer, et al. ACS Syn Bio. 2023 | ||
| yMM1763 (BY4741 Matα ura3Δ0::5' Ura3 homology, pRPL18B-Gal4DBD-CRY2(535)-tENO1, pRPL18B-Gal4AD-CIB1-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3' homology his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5) | Harmer, et al. ACS Syn Bio. 2023 | ||
| yMM1765 (BY4741 Matα ura3Δ0::5' Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagBM-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3' homology his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5) | Harmer, et al. ACS Syn Bio. 2023 | ||
| YPD Agar | SigmaAldrich | Y1500 |
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Cite This Article
Harmer, Z. P., McClean, M. N. High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro. J. Vis. Exp. (198), e65686, doi:10.3791/65686 (2023).