我们感谢与 Ju 实验室和中实验室的成员进行了深思熟虑的讨论, 以及莱特州立大学的财政支持。

注: 此处描述的协议用于筛选十96井板中包含的库质粒, 但可以相应地放大或缩小。需要重复该协议以完成整个图书馆的筛选。通常情况下, 每次对10块的图书馆基因进行筛查, 可以得到1人的舒适处理。
1. 96 井酵母转化剂的制备
注意: 此步骤按照前面描述的7、10进行。
<ol><li>整除5µL (约 50–100) 从阵列质粒库中的质粒 DNA 到圆底96井板的每一井。 注: 这样一个图书馆的例子是酵母基因过度表达图书馆10。</li>
	<li>接种150毫升的酵母蛋白胨葡萄糖 (YPD) 培养基, 在500毫升的烧瓶中有一个菌落 (2–3毫米直径) 的单倍体酵母菌株 W303α。一夜之间孵化他们在30°c 与震动 (200 转每分钟)。</li>
	<li>第二天早晨, 测量隔夜文化的 od600并且稀释酵母文化到 OD600 = 0.1 在 (至多) 2 L YPD。孵化它在30°c 与震动 (200 转每分钟) 为 ~ 5 h 直到文化到达 OD600 = 0.4–0.6。</li>
</ol>2. 酵母转化
<ol><li>通过填充8无菌250毫升离心机瓶和离心他们在室温下 3000 x g为10分钟来收获酵母培养. 在不破坏球团的情况下倒入上清液。 注: 在室温下执行所有离心步骤。</li>
	<li>用无菌蒸馏的 H2O 洗净酵母。为此, 增加100毫升不育 H2O 到每个离心机瓶和漩涡它并用重悬细胞颗粒。将洗涤过的细胞组合成2瓶, 并将其离心在 3000 x g处以5分钟。</li>
	<li>在每瓶100毫升的 0.1M LiOAc/1XTE (100 毫米 LiOAc; 10 毫米三, pH 8.0; 1 毫米 EDTA) 中清洗每个瓶子中的细胞, 并将其离心在 3000 x g上, 以5分钟为单位倒入上清。</li>
	<li>当离心, 煮沸5毫升鲑鱼精子 DNA (10 毫克/毫升) 在100°c 使用一个块加热器3分钟, 然后冷却它在冰上。</li>
	<li>并用重悬细胞颗粒在每瓶25毫升 0.1M LiOAc/1XTE, 结合悬浮细胞和转移到一个150毫升烧瓶。添加5毫升的预冷鲑鱼精子 DNA 和孵化它在30°c 与震动 (225 rpm) 30 分钟。</li>
	<li>将细胞混合物倒入无菌的一次性试剂库中, 使用多通道吸管, 将细胞混合物的35µL 转移到含有库质粒 DNA 的圆底96井板的每个井中。涡流96井板使用一个板块 vortexer 1 分钟在 1000 rpm。在30摄氏度的情况下孵育30分钟, 不摇晃。 注意: 不要堆叠板, 这样热量就能更有效地转移。我们发现, 涡流的96井板在 1000 rpm 不会导致液体溢出的油井, 但一个安全的涡流速度应该在执行步骤之前测试。</li>
	<li>在烧瓶中, 准备200毫升的转换缓冲器, 其中包含最终浓度为 40% PEG3350, 10% 亚砜和 0.1M LiOAc。在使用前立即准备转换缓冲器, 并通过摇动彻底混合。</li>
	<li>从30°c 孵化器中删除96井板, 并将其混合三十年代在 1000 rpm 使用板 vortexer。将转换缓冲器的125µL 添加到每个井中, 然后在1000转每分钟旋转1分钟的平板。</li>
	<li>孵化的板块在30°c 30 分钟, 然后热休克酵母通过放置在42°c 孵化器的15分钟。 注意: 不要堆叠板。</li>
	<li>在 3000 x g上离心板5分钟。通过在垃圾桶上反转板块, 并强行从盘子中倾倒缓冲物, 从油井中取出转换缓冲器。迅速将倒置的盘子涂抹在干净的纸巾上, 以除去盘子顶部的液体。</li>
	<li>将细胞添加到200µL 的最小脱落介质中, 相应于库质粒的可选标记。 注: 我们使用了一个综合市建局-媒介。</li>
	<li>将板材离心5分钟, 在 3000 x g处, 在垃圾桶上取出上清液, 如步骤2.10 所明。</li>
	<li>添加160µL 的最小的市建局-培养基含2% 葡萄糖。涡流板1分钟在 1000 rpm 和孵化他们在30°c 48 小时, 而不动摇。48小时后, 请注意, 在每个井的底部可见一个转化后的酵母颗粒。</li>
	<li>使用液体分配器, 添加100µL 的市建局-媒体含有葡萄糖的每一个新的一套96井板。</li>
	<li>涡流的板块包含转化酵母 (从步骤 2.13) 在1000转每分钟三十年代. 使用无菌塑料96针复制器, 将引脚放入含有转化酵母的井中, 然后将其接种到新板的相应井中。充满了媒体。孵化新的板材在30°c 为24小时。 注: 如果使用10板, 介质也可以用多通道吸管手动配药。</li>
	<li>24小时后, 每个井的底部都应可见一个酵母颗粒, 其中含有成功转化的酵母。为了保存这些酵母菌株作为甘油股票, 添加50µL 50% 甘油每一个井, 漩涡的板块为三十年代在 1000 rpm, 密封的盘子与密封胶带和冻结他们在-80 °c。 注意: 以上步骤只需要执行一次。不同酵母模型的未来筛选对同一图书馆一次性试剂库可以从甘油库存开始, 并立即从以下步骤进行。</li>
</ol>3. 含有图书馆基因的细胞与查询酵母的交配
<ol><li>要从甘油库存中恢复库株, 从-80 °c 冰箱取出96井板, 取出密封胶带, 让酵母在室温下解冻约30分钟。</li>
	<li>一旦酵母解冻, 使用无菌塑料96针复制器接种甘油股票到160µL 新鲜的市建局-媒体包含2% 葡萄糖在96井板和孵化他们在30°c 为 24 h. 在使用库菌株后立即, 用密封胶带封住盘子, 并将其退回到-80 摄氏度冰柜。</li>
	<li>在同一天图书馆菌株 (W303α) 解冻, 接种查询酵母菌株 (这里, FUS 在 W303a) 到50毫升 YPD 和增长它隔夜在30°c 与震动在250转每分钟。</li>
	<li>第二天早上, 将查询酵母菌倒入一个无菌的一次性试剂库, 用多通道吸管将查询菌株的160µL 整除到96井板的每一个井中。</li>
	<li>使用液体分配器, 将160µL 的 YPD 介质放入十96井板的每一个井中。使用这些板块以后交配的查询酵母菌株与图书馆酵母。</li>
	<li>简要地将含有查询应变的96井板涡旋, 然后使用无菌96针复制器将查询应变转移到 YPD 板上。</li>
	<li>简要地涡旋的图书馆应变板, 并为每个图书馆的应变板, 使用新的无菌96针复制器转移到 YPD 板, 已接种的查询应变。 注: 甘油细胞在多次解冻冷冻循环后失去活力。尽快将图书馆的应变恢复到长期储存 (-80 °c 冷冻库), 使图书馆保持良好的质量, 并准备好将来使用。适当维持, 甘油的库存可以冻结和解冻至少10倍。我们还强烈建议保存甘油库存的工作副本和备份副本。当工作库存不工作时, 立即从备份副本中制作新的工作副本。</li>
	<li>孵化的 YPD 板在30°c 24 小时, 请注意, 酵母颗粒将在每井底部可见24小时后。</li>
	<li>填充96井板, 其最小的脱落介质含有2% 糖, 对应于查询菌株中质粒的可选择标记以及库质粒 (例如, 市建局-他的-)。</li>
	<li>使用无菌的96针复制器将酵母从 YPD 交配培养基转移到选择性培养基上。孵育96井板在30°c 为 48 h;只有有交配和形成双细胞的酵母细胞, 因此, 含有的查询质粒和库质粒将能够生长在这个媒介。2天后, 观察在油井底部可见颗粒。</li>
</ol>4. 斑点测定法
<ol><li>
在含糖的选择性介质中, 经过48小时的生长, 在琼脂盘上发现酵母。
	<ol>
<li>准备2套市建局-他的辍学板含有2% 琼脂, 使用透明聚苯乙烯板, 其中含有2% 半乳糖, 另一个含有2% 葡萄糖。</li>
		<li>涡96井板1分钟在1000转, 然后发现酵母到市建局-他的辍学板包含2% 半乳糖和2% 琼脂 (FUS 和图书馆基因被诱导) 和对市建局-他的辍学板包含2% 葡萄糖和2% 琼脂 (FUS 和图书馆基因压制) 使用机器人的识别机, 其中每个井的文化被发现到四份的琼脂板块 (即, 1 井的文化被发现到4斑点的琼脂板)。</li>
		<li>发现后, 让琼脂板块干燥, 然后把它们倒在30摄氏度孵化器。每24小时拍摄琼脂板块, 以记录一个更快/更多的酵母生长, 其中的毒性, 以查询菌株被拯救或记录一个较慢/少的增长, 其中对查询菌株的毒性加剧。将盘子孵化4天。 注: 每个井中的文化可以在1到1的琼脂板上发现, 如以前的基于变换的方法10所示。然而, 这里的1到4的发现 (可以方便地使用机器人识别机设置) 显著增加了检测的鲁棒性, 减少误报的数量。只有当所有4个来自同一井的菌落表现出相似的表型时, 才会考虑正面撞击。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个在酵母中执行质粒过度表达筛的协议, 将质粒库引入到酵母模型中。利用这种方法, 可以用同质粒库转化的同一株酵母, 筛选出多种神经退行性疾病蛋白毒性酵母模型。转化过程只需要进行一次, 然后用高效的酵母交配将质粒库引入到查询菌株中。该协议确实依赖于使用机器人设备来分配媒体和斑点酵母培养到琼脂板块。虽然可以在不使用机器人设备的情况下执行该协议, 但?...

酵母酵母菌在 1基础科学研究中得到广泛应用, 以了解与人类疾病直接相关的细胞过程。此外, 它被用作研究人类疾病相关蛋白的模型有机体, 例如与最常见的神经退行性疾病相关的蛋白质, 包括阿尔茨海默病、帕金森病、亨廷顿氏病和肌萎缩症。侧索硬化症 (ALS)2。酵母模型的优点是可以轻松地使用一个基因组范围的屏幕来识别与疾病相关蛋白的毒性相关的细胞通路, 从而对其毒性机制进行深入的研究。一个这样的屏幕被称为一个过度表达的图书馆屏幕, 其中5500酵母基因的排列库被转化为酵母模型, 以确定哪些基因可以修改毒性时抗原。这种筛选方法已成功地应用于多种神经退行性疾病相关蛋白的酵母模型中, 包括亨廷顿氏病的杭丁顿蛋白3、4、5 的α synuclein., Aβ阿尔茨海默氏病6, FUS 和 TDP-43 为 ALS7,8,9。虽然它通常是在高通量的方式10, 最劳动密集型的步骤的屏幕是单独转换5500酵母基因从阵列库。这一步必须在每次重复筛选时执行, 并且每当需要研究新建立的酵母模型时。找到一个更有效的方法来完成这项任务是很重要的。
酵母细胞可以稳定地存在于单倍体和双体形态中。单倍体细胞有两种相反的交配类型, 交配型 a 和α. 每种交配类型的单倍体细胞产生和分泌它们自己特定的交配信息素, 只有相反的交配型细胞反应。这允许在 a 和α细胞之间交配产生稳定的双倍细胞, a/α。这个过程是自发和高效率的11。我们可以利用这种独特的酵母生命周期来介绍质粒库。更具体地说, 阵列质粒库中的每个基因都将转化为一个交配型的单倍体细胞,即α细胞。这些含有库基因的细胞将会以96井格式排列的甘油储存。对于每个需要筛选的酵母模型, 含有该库基因的酵母细胞可以从甘油类中解冻, 通过与交配类型的感兴趣的酵母模型交配来进行筛选,即交配型 a。这种使用交配把两个基因放到酵母中的想法并不是新的。它已成功地应用于高通量酵母双杂交筛选中, 其中一个交配类型的诱饵构造 (即Gal4-DNA-binding 域融合) 通过与一个阵列库的猎物构造交配而聚集在一起。12. 然而, 这一战略从来没有被应用于过度表达的图书馆放映中, 因为它一直采用传统的转换方法。
我们的实验室以前建立了一个酵母模型的 ALS 相关蛋白 FUS7。通过使用转换方法进行过表达库筛选, 我们发现了五种酵母基因 (ECM32、NAM8、SBP1、SKO1和VHR1), FUS 时抢救抗原的毒性。这些发现被独立地证实了以相似的研究由另一组8。hUPF1, 人类同源的ECM32, 后来被证明抑制毒性的主要神经细胞13和在动物模型 ALS14以及。利用这五种基因作为原理证明, 我们证明, 在 FUS 酵母模型中, 通过交配, 所有的五种基因都类似地拯救了 FUS 毒性。由于含有库基因的酵母细胞可以永久储存在甘油库存中, 需要时恢复, 这种基于交配的方法将在每次需要筛选库时消除耗时的转换步骤。由于交配效率高, 不涉及质粒的转化, 这一策略也大大降低了大型质粒库的纯化和转化所带来的成本。我们将成功地将此方法应用于 FUS 酵母模型的图书馆筛选。
图 1简要介绍了基于交配的筛查程序。最初, 阵列质粒库被转化成一个单倍体酵母菌株的交配型α使用高通量酵母转化协议, 其中每个井的96井板含有酵母转化为特定的图书馆质粒。这种转化酵母的收集保存为甘油的股票, 可以解冻和恢复使用后。酵母模型的兴趣, 在这种情况下, FUS 毒性, 必须产生的单倍体酵母菌株与相反的交配类型 (交配型 a)。利用无菌96针复制器的高通量方式, 将含有质粒库的 FUS 菌株和酵母菌转移到含有丰富培养基的96井板上, 并允许交配。交配后, 每一个井的交配培养的小体积转移到96井板含有合成的辍学媒体, 其中只有双酵母含有 FUS 和图书馆的基因可以增长。然后利用机器人识别机将各井的酵母培养物转移到琼脂板上, FUS 和库基因的表达。 此外, 酵母培养被发现控制琼脂板块在那里 FUS 和图书馆基因没有表达。继琼脂板块生长后, 会发现拯救或加剧 FUS 毒性的基因。

<table><tbody><tr><td>salmon Sperm DNA (SS-DNA)</td><td>Sigma-Aldrich</td><td>&amp;nbsp; D1626</td><td></td></tr><tr><td>YPD broth</td><td>Research Products International (RPI)</td><td>Y20090</td><td></td></tr><tr><td>Granulated Agar</td><td>Fisher Sci</td><td>BP97445</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Research Products International (RPI)</td><td>G32040</td><td></td></tr><tr><td>D-(+)-Galactose</td><td>Research Products International (RPI)</td><td>G33000</td><td></td></tr><tr><td>D-(+)-Raffinose Pentahydrate</td><td>Research Products International (RPI)</td><td>R20500</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Fisher Sci</td><td>A702-500</td><td></td></tr><tr><td>Synthetic Ura- drop out medium</td><td>Clontech</td><td>630416</td><td></td></tr><tr><td>Yeast amino acid drop out supplement -Histidine/-Uracil</td><td>Clontech</td><td>630422</td><td></td></tr><tr><td>Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate</td><td>Research Products International (RPI)</td><td>Y20060</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Sci</td><td>S67496</td><td></td></tr><tr><td>Lithium acetate, anhydrous</td><td>Fisher Sci</td><td>AC268640010</td><td></td></tr><tr><td>Polyethylene Glycol 3350 (PEG-3350)</td><td>Spectrum Chemical&amp;nbsp;</td><td>PO125-12KG</td><td></td></tr><tr><td>96 Pin Replicator&amp;nbsp;</td><td>Scinomix</td><td>SCI-5010-OS</td><td></td></tr><tr><td>Nunc OmniTray</td><td>Thermo Sci</td><td>140156</td><td></td></tr><tr><td>Corning Costar 96 well assay plate, round bottom with lid</td><td>Fisher Sci</td><td>07-200-760</td><td>non-treated, sterile</td></tr><tr><td>Eppendorf&amp;nbsp;Research plus Multichannel Pipette</td><td>Eppendorf</td><td>TI13690052</td><td>30-300ul volume</td></tr><tr><td>Fisherbrand&amp;nbsp;Isotemp Digital Dry Baths/Block Heaters</td><td>Fisher Sci</td><td>88-860-023</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;MixMate</td><td>Eppendorf</td><td>21-379-00</td><td></td></tr><tr><td>Eppendorf 5810R Centrifuge</td><td>Fisher Sci</td><td>05-413-112</td><td></td></tr><tr><td>Avanti J-26 XPI Centrifuge</td><td>Beckman</td><td>393127</td><td></td></tr><tr><td>MultiFlo FX Multi-Mode Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Rotor HDA&amp;nbsp;&amp;nbsp;</td><td>Singer Instruments</td><td></td><td></td></tr></tbody></table>

mating-based overexpression library screening in yeast

发芽酵母作为研究与人类疾病相关的蛋白质的模型被广泛应用。全基因组基因筛选是酵母研究中常用的有力工具。一些神经退行性疾病相关蛋白在酵母中的表达导致细胞毒性和聚集形成, 综述发现这些疾病的患者。在这里, 我们描述了一种筛选肌萎缩侧索硬化相关蛋白 FUS 酵母模型的方法, 以修饰其毒性。这个新的筛选平台不是使用转换, 而是依赖于酵母的交配来引入酵母模型中的粒度排列库。这种交配方法有两个明显的优点: 一是高效;二是将质粒预转换阵列库长期保存为甘油库存, 并可快速应用于其它筛网, 而不需要将劳动密集型的步骤转化为酵母模型。我们展示了这个方法如何能够成功地用于筛选基因, 改变 FUS 的毒性。

ALS 相关蛋白 FUS, RNA/DNA 结合蛋白, 以前研究的单倍体酵母7,8。遗传筛选采用基于转化的方法发现了几种抑制 FUS 毒性的酵母基因。其中一个酵母基因的人同源, 后来被证明是有效的抑制毒性的主要神经元细胞和大鼠模型的 ALS13。在这里, 我们使用相同的酵母模型表明, 过度表达的图书馆筛选可以通过交配有效地进?...

Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

Mating-based Overexpression Library Screening in Yeast

本文提出了一种基于交配的方法, 利用阵列质粒库, 促进萌芽酵母的超表达筛查。

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 ...

mating-based-overexpression-library-screening-in-yeast

Research

JoVE Journal

Genetics

7.6K 次观看.  Wright State University. 本文提出了一种基于交配的方法, 利用阵列质粒库, 促进萌芽酵母的超表达筛查。

视频: Mating-based Overexpression Library Screening in Yeast

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

科研

生物化学

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

遗传学

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫与感染

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, ...

生物学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

<meta charset="utf8"></head><body>通过酵母表面显示技术优化蛋白质功能

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

<meta charset="utf8"></head><body>通过 Yeast Surface Display 进行蛋白质工程

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

通过 Yeast Surface Display 进行蛋白质工程

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

生物工程

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

基于交配的酵母超表达文库筛选

摘要

探索更多视频

此视频中的章节

酵母 2-混合筛在批次中比较蛋白质相互作用

一种在裂变酵母中的深度固序辅助自发抑制屏

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

高通量酵母质粒过表达屏幕

条形码酵母图书馆竞争力的基因屏幕

通过 Yeast Surface Display 进行蛋白质工程

通过 Yeast Surface Display 进行蛋白质工程

通过 Yeast Surface Display 进行蛋白质工程

定向进化法