Name: directed evolution method in saccharomyces cerevisiae: mutant library creation and screening
Uploaded: 2016-04-01T13:00:00.000+00:00
Duration: 10 min 50 s
Description: Watch this Scientific Journal Video about Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening at JoVE.com

This work was supported by the European Commission project Indox-FP7-KBBE-2013-7-613549; a Cost-Action CM1303-Systems Biocatalysis; and the National Projects Dewry [BIO201343407-R] and Cambios [RTC-2014-1777-3].

1突变体库建设<ol><li>选择的区域要进行与基于所述可用的晶体结构或同源性模型18的计算算法的帮助下变形。 <ol><li>这里，目标从杏鲍菇 AAO的两个区域为随机诱变和重组（蛋氨酸[α1] -Val109，Phe392-Gln566），而扩增基因（844碱基对）的通过高保真PCR，其余（ 图1）。 注意:几个片段可以通过以独立或组合方式16变形进行研究。 </li></ol></li><li>通过诱变PCR扩增目标区域。创建通过叠加所定义的区域的PCR反应重叠区段之间的区域（〜各50碱基对）。 <ol><li>在含有DNA模板50微升（0.92纳克/微升），90纳米寡感（RMLN为段MI和AAO-BP的段M-II）的最终体积准备有针对性的细分诱变PCR，90纳米ntisense引物（AAO-92C为段MI和RMLC为段的M-II）中，0.3毫摩尔的dNTPs（0.075毫每个），3％（体积/体积）二甲基亚砜（DMSO），1.5毫氯化镁 ，0.05毫的MnCl 2和0.05 U /μLTaq DNA聚合酶。引物的序列在图1中详细说明。 </li><li>使用下述PCR程序:95℃2分钟（1个循环）; 95℃45秒，50℃45秒，74℃45秒（28个循环）;和74℃下进行10分钟（1个循环）。 </li></ol></li><li>放大用超高保真聚合酶的非诱变的区域，并包括重叠诱变区段和/或线性化载体突出的相应的区域。 <ol><li>在含有50微升的最终体积制备反应混合物:DNA模板（0.2纳克/微升），250纳米寡感HFF，250纳米寡聚反义HFR，0.8毫的dNTPs（每种0.2mM），3％（体积/体积）二甲基亚砜（DMSO）和0.02单位/微升iproof DNA聚合酶。引物序列在详述&lt;STRONG&gt;图1。 </li><li>使用下述PCR程序:98℃30秒（1次循环）; 98℃下10秒，55℃25秒，72℃45秒（28个循环）;和72℃10分钟（1个循环）。 注意:随着1.2描述和条件，1.3 43基点（质粒-M1区）的重叠; 46基点（M1区域-HF区）; 47碱基对（HF区-M2区）和61碱基对（M2 region-质粒）设计（ 图1）有利于在体内剪接在酵母。 </li><li>净化所有根据制造商的协议的商业凝胶提取试剂盒对PCR片段（诱变和非诱变）。 </li></ol></li><li>线性化使得侧翼大约50碱基对区域所创建的是同源的靶基因的5&#39;-和3&#39;-末端的载体中。 <ol><li>制备含2微克DNA，7.5ū 巴姆 HI，7.5ūXhoⅠ位 ，缓冲巴的20微克BSA和2微升线性反应混合物米的HI 10倍在20微升的最终体积。 </li><li>孵育反应混合物在37℃下2小时和40分钟。之后，继续进行灭活在80℃下进行20分钟。 </li></ol></li><li>净化用琼脂糖凝胶提取的线性化载体，以避免与残余环形质粒（ 图2）的污染。 <ol><li>在相邻的以及等分试样（5微升）在反应混合物的以及记者:加载消化反应混合物成半制备低熔点琼脂糖凝胶的大型孔（ⅴ0.75％，重量）。 </li><li>运行DNA电泳（电极之间的5伏/厘米，4ᵒC）和独立的对应大型井琼脂糖凝胶并将其存储在1X TAE 4ᵒC。 </li><li>色斑与分子量梯子，记者车道。可视化在紫外灯下条带。尼克的位置，其中线性载体的地方。 注意:作为纯化线性化载体的质量是一个criti校准因子在酵母重组成功和组装，避免半制备DNA电泳凝胶染色。使用染料和UV曝光对凝胶提取的可能影响DNA载体的稳定性，降低的体内重组效率。作为替代有毒的EtBr染料，凝胶红色和SYBR染料通常用于凝胶染色。 </li><li>在没有紫外线的光的，用在染色记者车道刻痕的指引，以便它可被分离鉴定在巨型井片段的线性化的载体。 </li><li>从琼脂糖提取的线性载体并用根据制造商的协议的商业凝胶提取试剂盒纯化它。 注意:使用高拷贝与抗生素和营养缺陷型标记附加型穿梭载体:在本例中，我们所用的尿嘧啶独立和氨苄青霉素抗性pJRoC30载体，酵母GAL1启动子的控制之下。 </li></ol></li><li>准备等摩尔英里PCR片段的夹具，并将其与在2线性化的载体混合:1的比例，不低于100纳克线性质粒（摩尔库/矢量开放测试不同比例达到良好的转化率）。 <ol><li>测量的PCR片段，并在260nm和280nm线性载体的吸光度以确定其浓度和纯度。 </li></ol></li><li>根据制造商的说明转化酵母感受态细胞，使用市售酵母转化试剂盒的DNA混合物（ 见表用品）。 <ol><li>在这里，使用缺陷的蛋白酶和URA3 -依赖S.酵母菌株，BJ5465。变换筛选（见下文）中与亲环化向量作为内部标准的细胞。此外，通过在不存在PCR片段转化线性载体检查的背景。 注:在检测最初的低分泌水平的情况下，使用S.像BJ5465酵母蛋白酶缺陷菌株，以促进活性的蛋白质的培养上清中的积累。如果目标酶经受超糖，使用糖基化缺陷型菌株（例如，Δkre2即仅能够连接小甘露糖的低聚物）可以是一个合适的选择。 </li></ol></li><li>板上的SC落出板中的转化的细胞，并培育它们在30℃下三天。板（上辅以尿嘧啶SC辍学板）URA3 - S.酵母细胞缺乏质粒作为用于筛选的阴性对照（见下文）。 </li></ol> 2.高通量筛选测定（图3） <ol><li>填充无菌96孔板的适当数量的与50微升最小培养基（23板，以分析的2000个克隆的库），每孔用移液机器人的帮助。 </li><li>从SC-降板挑单个菌落，并将它们转移到96孔板。 <ol><li>在每个板，接种列号6与亲型作为内部标准，用URA3以及H1 - S.酵母细胞（在补充有尿嘧啶SC培养基）无质粒作为阴性对照。 注:嗯H1与补充尿嘧啶辍学媒体专门填补。没有细胞的空白孔含有介质也可以制备作为附加无菌控制。 </li></ol></li><li>盖上盖子的板块，并在封口膜包裹其中。孵育板在30℃下48小时，225转和80％相对湿度的潮湿摇动。 </li><li>去除封口膜，添加160微升表达培养基到每个孔与移液机器人的帮助下，重新密封板并孵育他们另外24小时。 注:其他地方19日报道基本培养基和表达中做好准备。分泌水平可能取决于所研究的基因变化，并且相应地，叔他的温育时间，必须在每一种情况下进行优化以细胞生长在所有的孔中同步。 </li><li>离心10分钟2800×g下板（主平板）在4℃下。 </li><li>转移20微升从使用液体处理机器人多站中的主板到副本板孔中的上清液。 注意:为了有利于酶的分泌是可取由通常用于在酵母中（异源表达的信号肽替换目标蛋白质的天然信号肽例如，所述α因子前原领导者，来自酿酒在K 1杀手毒素的领导者酵母 ，甚至两种肽13）的嵌合版本。可替代地，天然信号肽可只进化为在酵母中的分泌。 </li><li>添加在100mM磷酸钠缓冲液pH 6.0 20微升2毫米毫米P -methoxybenzylalcohol与移液机器人的帮助。用96我们简要地搅动板LL板混合器，并培育他们在室温为30分钟。 </li><li>用移液机器人，添加160微升FOX试剂对每个副本板，并与在孔中的混合器（最终FOX混合物的浓度短暂搅拌:100μM的二甲酚橙，250μM的Fe（NH 4）2（SO 4）2和25mM 的 H 2 SO 4）。 <ol><li>几种添加剂加入试剂，以提高灵敏度，例如有机助溶剂（DMSO，乙醇，甲醇）或山梨糖醇17。这里，放大通过加入山梨糖醇至100mM（ 图4）的最终浓度的响应。 </li></ol></li><li>上读取板读数器在560nm的板（终点模式，叔0）。 </li><li>在室温下孵育板至显色，并再次测量吸收（叔1）。 <ol><li>从培养后的绝对值之间的差计算出相对活性和初始测量的归一化到的PAREntal类型每个板（ΔT1 - T0）。 </li></ol></li><li>主题的最佳突变命中两个连续的再检查，以排除假阳性。 注意:通常情况下，重新放映包括来自酵母，扩增和纯化在大肠杆菌质粒分离，接着鲜酵母细胞的转化用质粒19。每个选定克隆再筛选pentaplicate。 </li></ol>

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作者什么都没有透露。

在这篇文章中，我们总结了大部分的技巧和窍门在我们的实验室中通过S.定向进化工程师使用的酶酵母 （使用氧化铝作为一个例子），以便它们可以通过简单地按照此处描述的常用的方法适于使用与许多其它真核酶系统。 在库创建而言，变形是一个快速单罐法引进和重组中的小蛋白伸展随机突变，同时留下未改变16蛋白质的剩余区域。与几个突变负载库?...

定向分子进化是设计酶1,2健壮的，快速和可靠的方法，通过迭代轮次的随机突变，重组和筛选，可以产生改进的酶的版本的作用于新底物，在新颖的反应，在非天然的环境中，或甚至以协助细胞来实现新的代谢目标3-5。间在定向进化中使用的宿主，啤酒酵母酿酒酵母提供了不在原核生物同行6,7-否则可用复杂的真核蛋白质的功能性表达的解决方案的一个曲目。 在细胞生物学研究中详尽使用时，该小的真核模型在翻译后修饰，易于操纵和转换效率，所有这一切都是由定向进化8工程师酶重要性状方面有许多优点。此外，高频率在S同源DNA重组酵母加上其高效的校对设备打开一个广阔的图书馆创建和组装的基因在体内的可能性阵列，促进由单一的酶来复杂的人工途径9-12不同系统的演变。我们的实验室已经度过了过去十年的设计工具和策略在酵母中不同的木质素酶的分子进化（天然木材腐烂过程中参与木质素的降解氧化还原酶）13-14。在这种沟通中，我们提出了详细的方案编制和S.屏幕突变体文库酵母为一个模型flavooxidase， -芳基醇氧化酶（AAO 15） - ，可以很容易地转换为许多其它的酶。该协议涉及的聚焦定向进化方法:通过酵母细胞装置16，辅助（MORPHING同源体内编组诱变有组织重组处理）基于以检测分泌到培养液中17 AAO活性Fenton反应哒非常敏感筛选测定。

<table><tbody><tr><td>&lt;strong&gt;1. Culture media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A0166</td><td>CAS N&amp;ordm; 69-52-3 M.W. 371.39</td></tr><tr><td>Bacto Agar</td><td>Difco</td><td>214010</td><td></td></tr><tr><td>Cloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td>CAS N&amp;ordm; 56-75-7 M.W. 323.13</td></tr><tr><td>D-(+)-Galactose</td><td>Sigma-Aldrich</td><td>G0750</td><td>CAS N&amp;ordm; 59-23-4 M.W. 180.16</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>CAS N&amp;ordm; 50-99-7 M.W. 180.16</td></tr><tr><td>D-(+)-Raffinose pentahydrate</td><td>Sigma-Aldrich</td><td>83400</td><td>CAS N&amp;ordm; 17629-30-0 M.W. 594.51</td></tr><tr><td>Peptone</td><td>Difco</td><td>211677</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P0662</td><td>CAS N&amp;ordm; 7778-77-0 M.W. 136.09</td></tr><tr><td>Uracil</td><td>Sigma Aldrich</td><td>U1128</td><td></td></tr><tr><td>Yeast Extract</td><td>Difco</td><td>212750</td><td></td></tr><tr><td>Yeast Nitrogen Base without Amino Acids</td><td>Difco</td><td>291940</td><td></td></tr><tr><td>Yeast Synthetic Drop-out Medium Supplements without uracil</td><td>Sigma-Aldrich</td><td>Y1501</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. PCR Reactions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>dNTP Mix</td><td>Agilent genomics</td><td>200415-51</td><td>25 mM each</td></tr><tr><td>iProof High-Fidelity DNA polymerase</td><td>Bio-rad</td><td>172-5301</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M8054</td><td>CAS N&amp;ordm; 13446-34-9 M.W. 197.91</td></tr><tr><td>Taq DNA Polymerase</td><td>Sigma-Aldrich</td><td>D4545</td><td>For error prone PCR</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Plasmid linearization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BamHI restriction enzyme</td><td>New England Biolabs</td><td>R0136S</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>XhoI restriction enzyme</td><td>New England Biolabs</td><td>R0146S</td><td></td></tr><tr><td>Not I restriction enzyme</td><td>New England Biolabs</td><td>R0189S</td><td></td></tr><tr><td>Gel Red</td><td>Biotium</td><td>41003</td><td>For staining DNA</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;4. FOX assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonium iron(II) sulfate hexahydrate</td><td>Sigma-Aldrich</td><td>F3754</td><td>CAS N&amp;ordm; 7783-85-9 M.W. 392.14</td></tr><tr><td>Anysil Alcohol</td><td>Sigma Aldrich</td><td>W209902</td><td>CAS N&amp;ordm; 105-13-5 M.W. 138.16</td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td>CAS N&amp;ordm; 50-70-4 M.W. 182.17</td></tr><tr><td>Hydrogen peroxide 30%</td><td>Merck Millipore</td><td>1072090250</td><td>FOX standard curve</td></tr><tr><td>Xylenol Orange disodium salt</td><td>Sigma-Aldrich</td><td>52097</td><td>CAS N&amp;ordm; 1611-35-4 M.W. 716.62</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;5. Agarose gel stuff&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Norgen</td><td>28035</td><td>CAS N&amp;ordm; 9012-36-6</td></tr><tr><td>Gel Red</td><td>Biotium</td><td>41003</td><td>DNA analysis dye</td></tr><tr><td>GeneRuler 1kb Ladder</td><td>Thermo Scientific</td><td>SM0311</td><td>DNA M.W. standard</td></tr><tr><td>Loading Dye 6x</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>Low-melting temperature agarose</td><td>Bio-rad</td><td>161-3112</td><td>CAS N&amp;ordm; 39346-81-1</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;6. Kits and cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>S. cerevisiae strain BJ5465</td><td>LGC Promochem</td><td>ATTC 208289</td><td>Protease deficient strain with genotype: MAT&amp;alpha; ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL</td></tr><tr><td>E. coli XL2-Blue competent cells</td><td>Agilent genomics</td><td>200150</td><td>For plasmid purification and amplification</td></tr><tr><td>NucleoSpin Gel and PCR Clean-up Kit</td><td>Macherey-Nagel</td><td>740,609,250</td><td>DNA gel extraction</td></tr><tr><td>NucleoSpin Plasmid Kit</td><td>Macherey-Nagel</td><td>740,588,250</td><td>Column miniprep Kit</td></tr><tr><td>Yeast Transformation Kit</td><td>Sigma-Aldrich</td><td>YEAST1-1KT</td><td>Included DNA carrier (Salmon testes)</td></tr><tr><td>Zymoprep yeast plasmid miniprep I</td><td>Zymo research</td><td>D2001</td><td>Plasmid extraction from yeast</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;7. Plates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well plates</td><td>Greiner Bio-One</td><td>655101</td><td>Clear, non-sterile, polystyrene (for activity measurements)</td></tr><tr><td>96-well plates</td><td>Greiner Bio-One</td><td>655161</td><td>Clear, sterile, polystyrene (for microfermentations)</td></tr><tr><td>96-well plate lid</td><td>Greiner Bio-One</td><td>656171</td><td>Clear, sterile, polystyrene (for microfermentations)</td></tr></tbody></table>

directed evolution method in saccharomyces cerevisiae: mutant library creation and screening

设计用于生物技术应用的酶时，涉及建设，克隆和突变体库的表情，加上高频同源DNA重组在体内的过程在酿酒酵母定向进化提供了许多吸引人的优点。在这里，我们提出了一个协议，以创建一个基于真菌芳基醇氧化酶（AAO）的例子来提高其总活性酵母和屏幕突变体库。两种蛋白质的片段，用随机突变和体内 DNA重组进行集中定向进化。的〜50碱基对的侧翼每段悬允许AAO融合基因的正确重新装配在一个线性化的载体引起全自主复制质粒。与功能AAO变种丰富突变体文库的S.筛选酵母上清与基于芬顿反应灵敏的高通量测定法。的一般过程图书馆建设S.酵母这里描述可容易地应用于演进许多其他的真核基因，避免额外的PCR反应， 在体外 DNA重组和连接步骤。

从AAO P.杏鲍菇是一种细胞外flavooxidase供给真菌过氧化物用H 2 O 2与开始攻击木质素。 AAO的两段被以变形，以提高其活性及其在S.表达受到关注，定向进化酵母 19。由S.窝藏外国酶无关酵母 ，当在酵母构建突变体文库涉及特定重叠区域的工程有利于片段和其克隆入线性化载体之间的拼接的最关键的问题。在当前的例子...

Watch this Scientific Journal Video about Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening at JoVE.com

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 ...

directed-evolution-method-saccharomyces-cerevisiae-mutant-library

Research

JoVE Journal

Biology

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

10.8K 次观看.  Institute of Catalysis, CSIC. We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae. 

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

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

科研

遗传学

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 ...

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 ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

生物学

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, ...

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

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

定向进化法

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基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

基于交配的酵母超表达文库筛选

一种在裂变酵母中的深度固序辅助自发抑制屏

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌

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

化学遗传相互作用的快速鉴定