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>

<ol>
	<li>J&#228;ckel, C., Hilvert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalysts+by+evolution.">Biocatalysts by evolution.</a> Curr. Opin. Biotechnol. 21 (6), 753-759 (2010).</li><li>Bornscheuer, U. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+third+wave+of+biocatalysis.">Engineering the third wave of biocatalysis.</a> Nature. 485 (7397), 185-194 (2012).</li><li>Renata, H., Wang, Z. W., Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+enzyme+universe:+accessing+non-natural+reactions+by+mechanism-guided+directed+evolution.">Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.</a> Angew. Chem. Int. Ed. 54 (11), 3351-3367 (2015).</li><li>Cobb, R. E., Chao, R., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution:+past,+present+and+future.">Directed evolution: past, present and future.</a> AIChE J. 59 (5), 1432-1440 (2013).</li><li>Abatemarco, J., Hill, A., Alper, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+metabolic+engineering+toolbox+with+directed+evolution.">Expanding the metabolic engineering toolbox with directed evolution.</a> Biotechnol. J. 8 (12), 1397-1410 (2013).</li><li>Pourmir, A., Johannes, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution:+selection+of+the+host+organism.">Directed evolution: selection of the host organism.</a> Comput Struct Biotechnol J. 2 (3), e201209012 (2012).</li><li>Krivoruchko, A., Siewers, V., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+for+yeast+metabolic+engineering:+lessons+from+synthetic+biology.">Opportunities for yeast metabolic engineering: lessons from synthetic biology.</a> Biotechnol J. 6 (3), 262-276 (2011).</li><li>Gonzalez-Perez, D., Garcia-Ruiz, E., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+in+directed+evolution:+an+efficient+tool+to+improve+enzymes.">Saccharomyces cerevisiae in directed evolution: an efficient tool to improve enzymes.</a> Bioeng Bugs. 3, 172-177 (2012).</li><li>Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+protocols+in+Saccharomyces+cerevisiae+by+In+Vivo+Overlap+Extension.">Mutagenesis protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension.</a> Methods Mol. Biol. 634, 3-14 (2010).</li><li>Bulter, T., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+libraries+in+Saccharomyces+cerevisiae.">Preparing libraries in Saccharomyces cerevisiae.</a> Methods. Mol. Biol. 231, 17-22 (2003).</li><li>Ostrov, N., Wingler, L. M., Cornish, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+assembly+and+combinatorial+libraries+in+S.+cerevisiae+via+reiterative+recombination.">Gene assembly and combinatorial libraries in S. cerevisiae via reiterative recombination.</a> Methods. Mol. Biol. 978, 187-203 (2013).</li><li>Shao, Z., Zhao, H., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+assembler,+an+in+vivo+genetic+method+for+rapid+construction+of+biochemical+pathways.">DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways.</a> Nucleic Acids Res. 37 (2), e16 (2009).</li><li>Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+ligninolytic+enzyme+consortium.">Engineering the ligninolytic enzyme consortium.</a> Trends Biotechnol. 33 (3), 155-162 (2015).</li><li>Garcia-Ruiz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+ligninolytic+oxidoreductases:+from+functional+expression+to+stabilization+and+beyond.">Directed evolution of ligninolytic oxidoreductases: from functional expression to stabilization and beyond.</a> Cascade Biocatalysis: integrating stereoselective and environmentally friendly reactions. , 1-22 (2014).</li><li>Hernandez-Ortega, A., Ferreira, P., Martinez, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+aryl-alcohol+oxidase:+a+peroxide-producing+flavoenzyme+involved+in+lignin+degradation.">Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation.</a> Appl. Microbiol. Biotechnol. 93 (4), 1395-1410 (2012).</li><li>Gonzalez-Perez, D., Molina-Espeja, P., Garcia-Ruiz, E., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenic+organized+recombination+process+by+homologous+in+vivo+grouping+(MORPHING)+for+directed+enzyme+evolution.">Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) for directed enzyme evolution.</a> PLoS One. 9, e90919 (2014).</li><li>Rhee, S. G., Chang, T., Jeong, W., Kang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Detection+and+Measurement+of+Hydrogen+Peroxide+Inside+and+Outside+of+Cells.">Methods for Detection and Measurement of Hydrogen Peroxide Inside and Outside of Cells.</a> Mol. Cells. 29 (6), 539-549 (2010).</li><li>Sebestova, E., Bendl, J., Brezovsky, J., Damborsky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+tools+for+designing+smart+libraries.">Computational tools for designing smart libraries.</a> Methods. Mol. Biol. 1179, 291-314 (2014).</li><li>Vi&#241;a-Gonzalez, J., Gonzalez-Perez, D., Ferreira, P., Martinez, A. T., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+directed+evolution+of+aryl-alcohol+oxidase+in+yeast+using+chimeric+signal+peptides.">Focused directed evolution of aryl-alcohol oxidase in yeast using chimeric signal peptides.</a> Appl. Environ. Microbiol. , (2015).</li><li>Gay, C., Collins, J., Gebicki, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroperoxide+Assay+with+the+Ferric-Xylenol+orange+Complex.">Hydroperoxide Assay with the Ferric-Xylenol orange Complex.</a> Anal. Biochem. 273 (2), 149-155 (1999).</li><li>Reetz, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalysis+in+organic+chemistry+and+biotechnology:+Past,+present,+and+future.">Biocatalysis in organic chemistry and biotechnology: Past, present, and future.</a> J. Am. Chem. Soc. 135 (34), 12480-12496 (2013).</li><li>Mate, D. M., Gonzalez-Perez, D., Mateljak, I., Gomez de Santos, P., Vicente, A. I., Alcalde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pocket+manual+of+directed+evolution:+Tips+and+tricks.">The pocket manual of directed evolution: Tips and tricks.</a> Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications. , .</li><li>Chao, R., Yuan, Y., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+DNA+assembly+technologies.">Recent advances in DNA assembly technologies.</a> FEMS Yeast Res. 15, 1-9 (2015).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>CAS N&#186; 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>C-0378</td>
			<td>CAS N&#186; 56-75-7 M.W. 323.13</td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>CAS N&#186; 59-23-4 M.W. 180.16</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>CAS N&#186; 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&#186; 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&#186; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>2. PCR Reactions</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&#186; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3. Plasmid linearization</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>Gel Red</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>For staining DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4. FOX assays</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&#186; 7783-85-9 M.W. 392.14</td>
		</tr>
		<tr>
			<td>Anysil Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>W209902</td>
			<td>CAS N&#186; 105-13-5 M.W. 138.16</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td>CAS N&#186; 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&#186; 1611-35-4 M.W. 716.62</td>
		</tr>
		<tr>
			<td>Agarose gel stuff</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Norgen</td>
			<td>28035</td>
			<td>CAS N&#186; 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&#186; 39346-81-1</td>
		</tr>
	
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5. Kits and cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae strain BJ5465</td>
			<td>LGC Promochem, Spain</td>
			<td>ATTC 208289</td>
			<td>Protease deficient strain with genotype: MAT&#945; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>6. Plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655101</td>
			<td>Clear, non-sterile, Polystyrene (for activity measurements)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655161</td>
			<td>Clear, sterile, Polystyrene (for microfermentations)</td>
		</tr>
		<tr>
			<td>96-well plate lid</td>
			<td>Greioner 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

Universidad de Cartagena UDEC

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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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 ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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.

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耦合检测监控蛋白复性

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