优化液体搬运机器人对拟南芥进行高通量前化学遗传学筛选的应用
Summary
在模型植物物种 (拟南芥) 上, 对合成小分子进行了高通量筛查。该协议是为液体处理机器人开发的, 它提高了正向化学遗传学筛的速度, 加速了新的小分子对植物生理学的发现。
Abstract
化学遗传学正越来越多地被用来解码植物中可能由于基因冗余或致死性而顽固于传统遗传学的特性。然而, 合成小分子具有生物活性的可能性很小;因此, 必须对数以千计的分子进行测试, 以找到感兴趣的。液体处理机器人系统的设计, 以处理大量的样本, 增加了一个化学图书馆可以筛选的速度, 除了最小化/标准化的错误。为了在拟南芥(南芥) 上实现一个5万小分子库的高通量前向化学遗传学屏幕, 开发了使用台式多通道液体处理机器人的协议, 要求最小技术人员参与。通过这些协议, 发现了3271个小分子, 导致可见的表型改变。1563种化合物导致短根, 1148 种化合物变色, 383 种化合物导致根毛和其他, 非分类, 改变, 177 化合物抑制发芽。
Introduction
在过去的20年中, 植物生物学领域的研究人员已经取得了长足的进步, 使用化学遗传学方法, 无论是正反两方面, 都提高了我们对细胞壁生物合成、骨架、激素合成和信号传递的理解,安歇, 发病机制, 嘌呤生物合成, 和膜贩运1,2,3,4,5。使用正向化学遗传学技术可以识别感兴趣的表型, 并允许研究人员了解特定过程的基因型基础。相反, 逆向化学遗传学寻找与预先确定的蛋白质目标6相互作用的化学物质。在植物生物学中, 拟南芥一直处于这些发现的前沿, 因为它的基因组是小的、映射的和注释的。它有一个短的世代时间, 并且有多个变种人或报告者线为方便辨认异常亚细胞机器7。
有两个主要瓶颈阻碍了正向化学基因筛的进展、初始筛选过程和确定感兴趣的化合物的目标8。提高小分子选择速度的主要帮助是使用自动化和自动化设备9。液体搬运机器人是处理大型小分子库的绝佳工具, 在生物科学 (10) 的推动进程中发挥了重要作用。此处提出的协议旨在缓解与筛选过程相关的瓶颈, 从而使生物活性小分子的识别速度加快.该技术为经营者减轻了劳动和时间负担, 同时也降低了对原理调查人员的经济成本。
到目前为止, 大多数化学库都在1万到2万种化合物之间进行了分析, 其中一些有多达15万个, 一些与70个9、11、12、13、14、15,16. 本文介绍的协议是在5万化合物的小分子库 (见材料表) 上实施的, 迄今在拟南芥上进行了较大的正向化学遗传学筛。这项议定书符合目前的趋势, 以提高效率和速度的前瞻性化学遗传学, 特别是因为它涉及到除草剂发现, 杀虫剂发现, 杀菌剂发现, 药物发现和癌症生物学17 ,18,19,20,21。虽然这项协议在这里与拟南芥一起实施, 但可以很容易地适应细胞培养, 孢子, 甚至在 96-, 384, 或1536孔板中液体培养基中的昆虫。由于其体积小, 拟南芥可在96井板中进行筛选。然而, 在水井中均匀地分配种子是一个挑战。手工播种是准确的, 但劳动密集型, 虽然有设备设计, 以分配种子到96井板, 他们是昂贵的购买。在这里, 我们展示了如何绕过这一步, 只是一个小的准确性损失。
这个方法的总目标是使筛选一个大化学图书馆反对拟南芥更加可管理, 不妥协准确性,通过使用液体搬运机器人。这种方法的使用提高了研究者的效率, 减少了完成初始稀释系列管理和随后的表型屏幕的时间, 允许在解剖显微镜下快速可视化样本, 并迅速新的生物活性小分子的鉴定。图 1在4个步骤中描述了此协议的关键结果。
图 1: 正向化学遗传学屏幕的整体工作流.要为4个关键步骤中的每一项详细描述的协议概述。1: 接收化学图书馆, 2: 制作稀释库, 3: 制作筛板, 4: 孵化和可视化筛板。请单击此处查看此图的较大版本.
Protocol
1. 创建稀释库 用手工标签625稀释库板, 确保它们与化学库中相应的印版相匹配。此外, 通过控制台驱动器将流量和流出流软管连接到多通道尖端洗涤自动材质定位器 (阿尔卑斯) (参见材料表)。 访问计算机并通过设备控制器与多通道尖端洗涤磷酸酶的连接打开洗涤泵以循环水。这将在协议结束时自动关闭。 装载, 用手, 堆垛机10的附加到堆放机的传送带, 按以下顺?…
Representative Results
在解剖显微镜下, 在筛选浓度的基础上, 通过添加小分子来准确、高效地表征表型的能力, 是这种方法在拟南芥上进行化学遗传学的最终目的.当所有5万种化合物被筛选时, 观察到的表型是多种多样的, 可以分成几个不同的类 (图 2)。图 3A-f描述了在解剖显微镜下低放大率观察到的表型的例子。某些表型提供了不?…
Discussion
该协议旨在帮助研究人员完成对拟南芥的化学遗传学筛选。我们从5万种化合物 (图 2和图 3) 的屏幕上提供了具有代表性的结果, 其中一个最大的前向化学遗传学屏幕, 在拟南芥上进行, 日期为9,13,23。使用液体搬运机器人, 使稀释库更高效, 筛选库生成, 提高了新化合物识别的速度和效?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢 Jozsef 鹳, 米切尔里士满, Jarrad Gollihue, 和安德烈-桑切斯的建设性和关键的讨论。莎琳博士为表型照片。该材料的基础是国家科学基金会根据1355438号合作协议所支持的工作。
Materials
| Keyboard | Local Provider | N/A | Used for protocol design and operating the Biomek FX |
| Mouse | Local Provider | N/A | Used for protocol design and operating the Biomek FX |
| Computer Screen | Local Provider | N/A | Used for protocol design and operating the Biomek FX |
| Computer | Local Provider | N/A | Used for protocol design and operating the Biomek FX |
| DIVERSet Diverse Screening Library | ChemBridge | N/A | Chemical library |
| Biomek Software | Beckman Coulter | N/A | Runs and designs the Biomek FX |
| Device Controller | Beckman Coulter | 719366 | Operates the water pump/tip washing station |
| Stacker Carousel Pendent | Beckman Coulter | 148240 | Manual operation of Biomek Stacker Carousel |
| Biomek Stacker Carousel | Beckman Coulter | 148520 | Rotary unit that houses all FX Stacker 10's |
| FX Stacker 10 | Beckman Coulter | 148522 | Elevator unit that houses components for screen |
| FX Stacker 10 | Beckman Coulter | 148522 | Elevator unit that houses components for screen |
| FX Stacker 10 | Beckman Coulter | 148522 | Elevator unit that houses components for screen |
| FX Stacker 10 | Beckman Coulter | 148522 | Elevator unit that houses components for screen |
| Biomek FX | Beckman Coulter | https://www.beckman.com/liquid-handlers | Robot that performs the desired operations |
| Accuframe | Artisan Technology Group | 76853-4 | Frames arm to place components corretly |
| Framing Fixture | Beckman Coulter | 719415 | Centers arm in the Accuframe |
| Multichannel Tip Wash ALP | Beckman Coulter | 719662 | Washes the tips after the ethanol bath |
| Tip Loader ALP | Beckman Coulter | 719356 | Pneumatically loads tips onto the arm |
| Air Compressor | Local Provider | N/A | Provides air for pneumatic tip loading |
| MasterFlex Console Drive | Cole-Parmer | 77200-65 | Pump used to circulate water through the Multichannel Tip Washer |
| Air Hose | Local Provider | N/A | Provides air from air compressor to Tip Loader |
| Water Hose | Local Provider | N/A | Provides water from 5 Gallon Reserviour to Tip Washer |
| Static ALP's | Beckman Coulter | Comes with Biomek FX | Supports equipment for the Screen |
| 5 Gallon Reserviour | Local Provider | N/A | Recirculates the dirty water from cleaning the tips |
| Grippers | Beckman Coulter | Comes with Biomek FX | Grabs and moves the equipment to the correct places |
| 96-Channel 200 µL Head | Beckman Coulter | Comes with Biomek FX | Holds the 96 tips used within the screen |
| AP96 P200 Pipette Tips | Beckman Coulter | 717251 | Used to make the screening library |
| 96 Well Flat Bottom Plate | Costar | 9018 | Aids in visulization of screen |
| 96 Well V-Bottom Plate | Costar | 3897 | Aids in storing of dilution library |
| AlumaSeal 96 Sealing Film | MedSci | F-96-100 | Seals for storage both the chemicle library and dilution library |
| Plastic ziplock sandwich bags | Local Provider | N/A | Used to ensure a humid environment for screen |
| AP96 P20 Pipette Tips | Beckman Coulter | 717254 | Used in the dilution library creation |
| Growth Chamber | Percival | AR36L3 | Germinates seeds for phenotypic visulization |
| Spatula | Local Provider | N/A | Holds seeds to add into wells where liquid seeding failed seed adequatly |
| Toothpick | Local Provider | N/A | Pushes seeds from spatula to wells |
| Murashige and Skoog Basal Salt Mixture | PhytoTechnology Laboratories | M524 | Add to MS media mixture |
| MES Free Acid Monohydrate | Fisher Scientific | ICN19483580 | Added to MS media to decrease pH |
| Agar Powder | Alfa Aesar | 9002-18-0 | Increases thickness of media to support seed suspension |
| 5M KOH | Sigma-Aldrich | 484016 | Increases pH to adequate levels |
| 1L Media Storage Bottle | Corning | 1395-1L | Holds enough media for a screen |
| Polypropylene Centrifuge Tubes | Corning | 431470 | Sterilizes seeds prior to vernilization |
| pH Probe | Davis Instruments | YX-58825-26 | Used for making media |
| ALPs (Automated Labware Positioners) Users Manual | Beckman Coulter | PN 987836 | Aids in setting up the accompaning equipment for the Biomek FX |
| Biomek 2000 Stacker Carousel Users Guide | Beckman Coulter | 609862-AA | Aids in setting up the Stacker Carousel |
| Biomek FX and FXP Laboratory Automation Workstations Users Manual | Beckman Coulter | PN 987834 | Used to frame the Multichannel Pod |
| Biomek FXP Laboratory Automation Workstation Customer Startup Guide | Beckman Coulter | PN B32335AB | Used to aid in setting up the Biomek FX |
| Biomek Software User's Manual | Beckman Coulter | PN 987835 | Used to set up and understand the Software |
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Citer Cet Article
Amos, B. K., Pook, V. G., Debolt, S. Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana. J. Vis. Exp. (134), e57393, doi:10.3791/57393 (2018).