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。

1. 创建稀释库
<ol><li>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)。</li>
	<li>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。</li>
	<li>装载, 用手, 堆垛机10的附加到堆放机的传送带, 按以下顺序在酒店 A D (图 4, 码垛);一盒 AP96 P20 吸管提示在房间 1, 四96井 V 底板的房间 2-5 与两个上部板块包含库存浓度从有序库和两个较低的板块空 (图 5, 堆垛机)。另外, 在房间6和四96井 V 底板上装载一盒 AP96 P20 吸管提示在 7-9 与二个上部板材包含有序的图书馆的存货集中和二个更低的板空 (图 5, 堆垛机)。</li>
	<li>通过手工设置, 甲板上有一个300毫升水水库在 P3, 一个300毫升70% 乙醇浴 P7, 尖端装载者阿尔卑斯 (TL1), 和多通道尖端洗涤阿尔卑斯 (TW1) (图 4, 甲板和图 5, 甲板)。</li>
	<li>使用操作软件, 现在 AP96 P20 吸管提示从堆垛机10和移动他们到尖端装载者阿尔卑斯。 注: 1.5 至1.12 均采用液体搬运机器人的操作软件完成;请参阅材料表。</li>
	<li>目前2房间从酒店 A 和分开所有四96井 V 底板在甲板上, 放置底部二在 P4 和 P8 和前二在 P5 和 P9 (图 4)。</li>
	<li>负载 AP96 P20 吸管提示与尖端装载者的磷酸酶到96通道200µL 头。从300毫升水油藏中抽出90µL, 并将 P4 上的96井 V 底稀释板抽出。对 P8 上的盘子重复此步骤。</li>
	<li>将化学库板混合在 P5 上, 反复抽吸和配药15µL 三次。另外, 从 P5 上的化学库板中抽出10µL, 并将10µL 放入 P4 的稀释板中。</li>
	<li>通过反复抽吸和配药50µL, 将 P4 板的溶液混合在一起, 共三次。一旦混合, 清洁 AP96 P20 吸管提示, 通过吸和配药70µL 70% 乙醇从 P7, 然后洗涤他们在多通道尖端洗涤磷酸酶通过吸和配药110% 水量四倍。</li>
	<li>对 P8 和 P9 上的第二对板重复步骤 1.8-1.9。创建第二个96井 V 底稀释板后, 按以下顺序从下到上堆叠板: P9、P5、P8 和 P4。然后, 把栈放在一个空的静止的高山上;无论是 P1、P2、P6、P10、P11、P12 或 P13。</li>
	<li>重复步骤1.6 至 1.10, 直到酒店 A 5 室为空。在到达6室时重复步骤 1.5, 将新的 AP96 P20 吸管提示移到尖端装载者的磷酸酶上, 并将所用的 AP96 P20 吸管提示放在空的静态碱性磷酸酶上。</li>
	<li>重复步骤1.6 至 1.10, 直到酒店 A 的9室为空。然而, 为了前往 B 酒店, 甲板上的盘子和小费必须重新装入酒店 A。</li>
	<li>用手重新填充300毫升的水水库。这一步是至关重要的, 计算机程序可以合并暂停详细说明此消息, 要求用户点击 "继续", 然后再执行下一步骤。</li>
	<li>重复步骤 1.5-1.13 为其余的酒店, 确保一个完整的300毫升水水库每次之前, 前往下一个酒店。</li>
</ol>2. 添加培养基-种子混合物筛板
<ol><li>通过添加4.3 克 MS 盐、0.50 克 MES、1.0 克琼脂至 1 L ½, 使 Murashige 和 Skoog (ms) 培养基与0.1% 琼脂结合, 在 ph 探针的监测下, 将 ph 值调整为 2, 但同时添加5.7 米氢氧化钾.</li>
	<li>用1% 的漂白剂和 SDS 在15和30分钟之间摇动种子, 然后用离心法冲洗4次, 用相同容积的水进行杀菌。一旦种子是无菌的, 将其放置在4摄氏度从24小时到7天为 vernilization。拟南芥生物资源中心描述了杀菌、vernilization 和生长的其他方法22。</li>
	<li>用手在密度为 0.1 g/100 毫升的培养基上添加种子。这个密度导致平均 3-10 种子每井96井板材。</li>
	<li>在酒店 a (图 6, 酒店 a) 的1和2房间中, 手工放置四96井平底板。将一盒 AP96 P250 吸管小贴士放在尖端装载机上, 一个300毫升的水库, 充满了在 P3 步骤 2.1-2.3 中创建的媒体种子混合物, 以及300毫升水库, 在 P7 (图 70%, 甲板和图 4上填充了6乙醇。, 甲板)。 注: 2.5 到2.8 是用操作软件完成的。</li>
	<li>目前客房1和2在酒店 A, 并分开的栈四板块。在每个空的静态阿尔卑斯 (P4、P5、P6、P8、P9、P10、P11 和 P12) 上放置一个盘子。负载 AP96 P250 吸管提示96通道200µL 头。</li>
	<li>从300毫升的 P3 介质种子库中抽出90µL, 并将其放入前96井平底板中。重复此过程, 直到所有八个板块包含介质种子混合物。</li>
	<li>通过从300毫升水库中吸出70µL, 并在 P7 上填充70% 乙醇, 清洁 AP96 P250 吸管提示。在多通道尖端洗涤磷酸酶的提示冲洗和配药110% 卷水四次, 卸载的提示在 TL1, 并收集的盘子手工。</li>
</ol>3. 将小分子添加到筛板上
<ol><li>手动装载, 一盒 AP96 P250 吸管提示进入酒店1室 a, 两个96井 V 底稀释库板进入房间 2, 4, 6 和 8, 和两个96井平底筛板进入房间 3, 5, 7 和 9 (图 4, 堆垛机和图 7, A 酒店)。此外, 连接软管到和从多通道尖端洗涤磷酸酶到5加仑的水库。 注: 3.2 到3.10 是用操作软件完成的。</li>
	<li>配置甲板, 以包含300毫升70% 乙醇洗涤水库在 P7;介质种子库可以放在 P3 的甲板上 (图 4、甲板和图 7、甲板)。另外, 通过设备控制器的连接打开控制台驱动器, 通过多通道尖端洗涤磷酸酶循环水。这将在协议结束时自动关闭。</li>
	<li>AP96 P250 吸管提示盒从酒店 A, 并将其移动到顶部装载者阿尔卑斯。</li>
	<li>提出96井 V 底稀释库板从2房间 A 到甲板和安置一个在静态阿尔卑斯 P4 和一个在 P8。目前96井平底筛板从酒店 A 3 室到甲板上, 并放置一个在静态的高山 P5 和一个在 P9。</li>
	<li>负载 AP96 P250 吸管提示与尖端装载者的磷酸酶到96通道200µL 头。</li>
	<li>将96井 V 底稀释板与 P4 50 µL 三倍的吸气和配药混合在一起。接着, 从这个盘子中抽出10µL, 并在 P5 上抽出96井平底筛板。</li>
	<li>将溶液在 P5 中混合, 通过吸气和配药50µL 三倍。用乙醇清洗 AP96 P250 吸管小贴士, 从 P7 的水库中吸出70µL 70% 乙醇, 然后在多通道尖端洗涤磷酸酶中通过吸气和分配110% 容积的水四次清洗。</li>
	<li>对第二个96井 V 底稀释库板 (P8) 和96井平底筛板 (P9) 重复步骤3.5 和3.6。</li>
	<li>将两个96井的 V 底稀释库板和两个96井平底筛板叠在一起。将板块移动到静态阿尔卑斯 P1、P2、P6、P10、P11、P12 或 P13。</li>
	<li>重复步骤 3.4-3.9 三次, 添加稀释的化学品, 以筛板共八次。最后, 通过视觉构象检查筛板每口井中种子的数量, 并通过额外的灭菌和 vernalized 的种子补充那些少于三种子的水井。</li>
</ol>4. 筛板的孵化和可视化
<ol><li>在干燥的容器中, 在16/8 光/暗循环中, 在22摄氏度的环境室中孵化96井平底筛板四天。在解剖显微镜下可视化96井平底筛板。记录所有异常表型以供进一步调查。</li>
</ol>

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作者声明他们没有竞争的财政利益。

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?...

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。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57393/57393fig1.jpg"> 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。<a href="/files/ftp_upload/57393/57393fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>Keyboard</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer Screen</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>DIVERSet Diverse Screening Library</td>
			<td>ChemBridge</td>
			<td>N/A</td>
			<td>Chemical library</td>
		</tr>
		<tr>
			<td>Biomek Software</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Runs and designs the Biomek FX</td>
		</tr>
		<tr>
			<td>Device Controller</td>
			<td>Beckman Coulter</td>
			<td>719366</td>
			<td>Operates the water pump/tip washing station</td>
		</tr>
		<tr>
			<td>Stacker Carousel Pendent</td>
			<td>Beckman Coulter</td>
			<td>148240</td>
			<td>Manual operation of Biomek Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek Stacker Carousel</td>
			<td>Beckman Coulter</td>
			<td>148520</td>
			<td>Rotary unit that houses all FX Stacker 10&#39;s</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>Biomek FX</td>
			<td>Beckman Coulter</td>
			<td>https://www.beckman.com/liquid-handlers</td>
			<td>Robot that performs the desired operations</td>
		</tr>
		<tr>
			<td>Accuframe</td>
			<td>Artisan Technology Group</td>
			<td>76853-4</td>
			<td>Frames arm to place components corretly</td>
		</tr>
		<tr>
			<td>Framing Fixture</td>
			<td>Beckman Coulter</td>
			<td>719415</td>
			<td>Centers arm in the Accuframe</td>
		</tr>
		<tr>
			<td>Multichannel Tip Wash ALP</td>
			<td>Beckman Coulter</td>
			<td>719662</td>
			<td>Washes the tips after the ethanol bath</td>
		</tr>
		<tr>
			<td>Tip Loader ALP</td>
			<td>Beckman Coulter</td>
			<td>719356</td>
			<td>Pneumatically loads tips onto the arm</td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air for pneumatic tip loading</td>
		</tr>
		<tr>
			<td>MasterFlex Console Drive</td>
			<td>Cole-Parmer</td>
			<td>77200-65</td>
			<td>Pump used to circulate water through the Multichannel Tip Washer</td>
		</tr>
		<tr>
			<td>Air Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air from air compressor to Tip Loader</td>
		</tr>
		<tr>
			<td>Water Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides water from 5 Gallon Reserviour to Tip Washer</td>
		</tr>
		<tr>
			<td>Static ALP&#39;s</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Supports equipment for the Screen</td>
		</tr>
		<tr>
			<td>5 Gallon Reserviour</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Recirculates the dirty water from cleaning the tips</td>
		</tr>
		<tr>
			<td>Grippers</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Grabs and moves the equipment to the correct places</td>
		</tr>
		<tr>
			<td>96-Channel 200 &#181;L Head</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Holds the 96 tips used within the screen</td>
		</tr>
		<tr>
			<td>AP96 P200 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717251</td>
			<td>Used to make the screening library</td>
		</tr>
		<tr>
			<td>96 Well Flat Bottom Plate</td>
			<td>Costar</td>
			<td>9018</td>
			<td>Aids in visulization of screen</td>
		</tr>
		<tr>
			<td>96 Well V-Bottom Plate</td>
			<td>Costar</td>
			<td>3897</td>
			<td>Aids in storing of dilution library</td>
		</tr>
		<tr>
			<td>AlumaSeal 96 Sealing Film</td>
			<td>MedSci</td>
			<td>F-96-100</td>
			<td>Seals for storage both the chemicle library and dilution library</td>
		</tr>
		<tr>
			<td>Plastic ziplock sandwich bags</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used to ensure a humid environment for screen</td>
		</tr>
		<tr>
			<td>AP96 P20 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717254</td>
			<td>Used in the dilution library creation</td>
		</tr>
		<tr>
			<td>Growth Chamber</td>
			<td>Percival</td>
			<td>AR36L3</td>
			<td>Germinates seeds for phenotypic visulization</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Holds seeds to add into wells where liquid seeding failed seed adequatly</td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Pushes seeds from spatula to wells</td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture</td>
			<td>PhytoTechnology Laboratories</td>
			<td>M524</td>
			<td>Add to MS media mixture</td>
		</tr>
		<tr>
			<td>MES Free Acid Monohydrate</td>
			<td>Fisher Scientific</td>
			<td>ICN19483580</td>
			<td>Added to MS media to decrease pH</td>
		</tr>
		<tr>
			<td>Agar Powder</td>
			<td>Alfa Aesar</td>
			<td>9002-18-0</td>
			<td>Increases thickness of media to support seed suspension</td>
		</tr>
		<tr>
			<td>5M KOH</td>
			<td>Sigma-Aldrich</td>
			<td>484016</td>
			<td>Increases pH to adequate levels</td>
		</tr>
		<tr>
			<td>1L Media Storage Bottle</td>
			<td>Corning</td>
			<td>1395-1L</td>
			<td>Holds enough media for a screen</td>
		</tr>
		<tr>
			<td>Polypropylene Centrifuge Tubes</td>
			<td>Corning</td>
			<td>431470</td>
			<td>Sterilizes seeds prior to vernilization</td>
		</tr>
		<tr>
			<td>pH Probe</td>
			<td>Davis Instruments</td>
			<td>YX-58825-26</td>
			<td>Used for making media</td>
		</tr>
		<tr>
			<td>ALPs (Automated Labware Positioners) Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987836</td>
			<td>Aids in setting up the accompaning equipment for the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek 2000 Stacker Carousel Users Guide</td>
			<td>Beckman Coulter</td>
			<td>609862-AA</td>
			<td>Aids in setting up the Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek FX and FXP Laboratory Automation Workstations Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987834</td>
			<td>Used to frame the Multichannel Pod</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation Customer Startup Guide</td>
			<td>Beckman Coulter</td>
			<td>PN B32335AB</td>
			<td>Used to aid in setting up the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek Software User&#39;s Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987835</td>
			<td>Used to set up and understand the Software</td>
		</tr>
	</tbody>
</table>

optimizing the use of a liquid handling robot to conduct a high throughput forward chemical genetics screen of arabidopsis thaliana

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。

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?...

Watch this Scientific Journal Video about Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana at JoVE.com

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

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

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。

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

optimizing-use-liquid-handling-robot-to-conduct-high-throughput

Research

JoVE Journal

Bioengineering

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

6.6K 次观看.  University of Kentucky. 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。

视频: Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

触觉机器人适应在3T功能磁共振成像

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA折纸设计生物反应灵敏的机器人

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...