这项工作是由以色列科学基金会的支持。

一酵母表达系统的设计III型效应校准适合III型效应（S）的利益表达的酵母系统的一项重要任务，可能需要一些试验和错误。和优化设计这样一个系统时，应考虑的主要相关因素有：1）启动子驱动表达的效应（S），2）效应基因的副本，3号）的抗原表位的标签，用来验证蛋白的表达和4）的酵母菌株。 1）启动由于细菌Ⅲ型效应可能对酵母细胞有毒性，应使用诱导启动子控制的表达。 GAL1启动子通常用于此目的，它的活动是在培养基中的碳源的规管：它是被压抑的葡萄糖和半乳糖诱导。但据报道，这个发起人轻微漏水，造成意外的毒性条件下压制。如果遇到类似的问题，最好是尽量减少效应基因的拷贝数如下所述，或使用替代诱导促销员，如MET3或CUP1促销员1 ，。在这里，我们描述为一个半乳糖诱导的启动子表达的效应蛋白的程序。 2）基因拷贝数蛋白表达水平的影响是一个额外的参数效应基因在细胞中的拷贝数。高表达水平的效应基因进行时2微米质粒（每单元40-60份）。中间表达式是通过使用每个细胞含有一个着丝粒质粒（1-3份），而低表达效应基因通过同源重组整合到酵母基因组。 “相结合的着丝粒含载体和GAL1推动者，我们已经成功地表达了约50植物病原黄单胞菌野油菜PV。vesicatoria和假单胞菌丁香光伏效应。免疫印迹分析检测的水平和抑制条件下可以忽略不计漏（所罗门和塞萨的番茄，未发表）。 3）抗原表位标记如果抗体对效应的利益都无法使用，抗原表位标记是融合的效应蛋白，免疫印迹，以监察其表达。常用的标签Myc基因，血凝素（HA）或标志，可靠的商业抗体。我们建议使用只有一个标签的副本，以最大限度地减少不受欢迎的效应蛋白的结构和活动的有害影响。 4）酵母菌株一个要表达的酵母菌株的根本要求是营养缺陷型表达载体的选择标记。重要的是要注意不同的酵母菌株，表达一定的效应可能会显示不同的敏感性。例如，我们观察到，BY4741和W303的菌株有几个油菜黄单胞菌PV。vesicatoria效应（所罗门和塞萨，未发表）不同的敏感性。因此，它是最好的测试效应（S）在不同的酵母菌株的兴趣。 二。酵母媒体的制备不包含任何载体，使用YPD（10 g / L酵母提取物，20 g / L的蛋白胨，2％W / V]葡萄糖）作为生长介质中的酵母菌株的生长。对于固体培养基，添加2％的解决方案（如果是非常柔软的固体培养基，添加0.05％[V / V]从5 N氢氧化钠原液中型高压灭菌前）[W / V]琼脂。我们建议准备无葡萄糖介质最终体积的90％和高压灭菌。不久之前使用，填补一个过滤器消毒的20％W / V]的葡萄糖溶液（20％葡萄糖溶液保持在4 ° C，以防止污染）的100％的体积。 酵母菌株含有一个载体，提供prototrophy选择标记（亮氨酸，尿嘧啶，组氨酸或色氨酸）的增长，使用不亮氨酸合成辍学的媒介，尿嘧啶，组氨酸和色氨酸（6.7 g / L酵母无氨基酸氮基，1.4 g / L酵母合成辍学中等补充）[W / V]葡萄糖或2％W / V]半乳糖和1％[W / V]棉子糖，含2％。对于固体培养基，添加2％W / V]琼脂的解决方案。我们建议准备在最终体积的90％的中等和高压灭菌。不久，在使用前，填写一个过滤器消毒的20％葡萄糖溶液，或过滤灭菌的半乳糖20％+ 10％棉子糖的解决方案取决于所需的碳源（保持20％的葡萄糖和20％，100％的体积半乳糖+ 10％棉子解决方案，在4 ° C，以防止污染）。根据选择标记的表达载体器，使用前（10毫升/ 1 g/100毫升亮氨酸股票大号，大号10毫升/ 200毫克毫升尿嘧啶股票，2毫升/ L，从1 g/100添加其他氨基酸或尿嘧啶毫升组氨酸的股票，或2毫升/升）从1 g/100毫升色氨酸股票。当准备亮氨酸和尿嘧啶的股票，通过高压灭菌消毒，并保持室温。筹备组氨酸和色氨酸股票时，通过过滤消毒，包装用铝箔，以防止光的瓶子，并保持在4 ° C。在程序所述，辅以与亮氨酸合成辍学介质，尿嘧啶，组氨酸和色氨酸被指定为合成完全培养基。 固体介质板可以存储长达两个月，在4 ° C使用前，在无菌的层流罩在室温为20分钟的干板。 三。酵母转化<ol><li>酵母转化过程开始之前，准备好以下三个解决方案：50％W / V]聚乙二醇（PEG）3350解决方案，1米LiAc溶液的pH值= 8.0，和TE溶液（100毫米的Tris pH值= 6.85 10 mM EDTA的pH值= 8.0）。过滤消毒的解决方案，并保持在4 ° C。 </li><li>使用长木轴或无菌接种环，挑一个酵母细胞集落（直径1-2毫米），并从一个全新的板块，在15毫升的聚丙烯管接种3毫升YPD。涡简要。培养管放置在滚筒和孵化在30℃不断旋转的隔夜。 </li><li>在早上，取出在室温下5分钟离心辊和800克的文化。离心后，彻底去除上清。 </li><li>重悬在1毫升的再悬浮缓冲液（10％[V / V] TE溶液，10％[V / V] 1米LiAc解决方案），由涡旋混合的细胞。 </li><li>对于每个要转化质粒和一个额外的控制改造，100μL的细胞悬液转移到微管。 </li><li>每管中，加入5μL单链鲑鱼精子DNA（10毫克/毫升）的质粒DNA转化（为控制管除外）和250-500纳克。 </li><li>认真添加到转换解决方案（10％，80％的50％[V / V] [W / V] PEG解决方案[V / V] TE溶液，10％[V / V] 1米LiAc解决方案的每管650μL ）和旋涡。 </li><li>在30 ° C下孵育30分钟滚筒。 </li><li>从摇床中取出试管，加入70μL的DMSO，反相管组合的10-15倍（不旋涡，以避免破坏细胞）。 </li><li>管放置在42 ° C预温水浴孵育15分钟。 </li><li>从水浴中取出试管，立即置于冰上2分钟。 </li><li>管一旦冷却下来，离心800克，在室温下5分钟。 </li><li>采取的离心管，并仔细清除粘性上清使用移液器。 </li><li>每管细胞沉淀重悬于150μL无菌双蒸水（DDW），传播一种人工合成的完全培养基含2％葡萄糖作为碳源板和无细胞悬液的氨基酸或核苷酸质粒提供prototrophy。 </li><li>离开板2分钟打开，让他们干层流罩。两三天的板放置在30℃培养箱。 </li><li>一旦菌落（直径1-2毫米）板块出现，挑选每个改造的约10个单菌落，他们转移到一个新的板块。当转移的殖民地，使2厘米× 2厘米的补丁，让大量的酵母细胞的增长。两天，在30 ° C孵育孵化器板。 </li><li>当酵母补丁已经长大，从孵化器中取出的板，并用封口膜密封。板可以被保存在4 ° C转移到一个新的板，每10-14天的殖民地。 </li></ol>四。准备酵母蛋白提取物，以验证免疫印迹效应表达<ol><li>选择适当的合成完整的与2％葡萄糖作为碳源的培养基中含有3毫升15毫升的聚丙烯管，从一个全新的板块没有的氨基酸或核苷酸的质粒的酵母殖民地（直径1-2毫米）提供选择prototrophy。培养管放置在滚筒和孵化在30 ° C过夜</li><li>在次日，从滚筒中删除的文化和800克离心机在室温下5分钟。取出离心管上清液。 </li><li>重悬细胞沉淀在3毫升无菌DDW和涡旋混合。 </li><li>再次离心，弃上清，悬浮细胞，在无菌DDW 3毫升。 </li><li> 200μL的细胞悬液转移到一个新的含有半乳糖2％和1％的棉子糖作为碳源的培养基中合成完整的3毫升15毫升管，没有的氨基酸或核苷酸，质粒的选择提供prototrophy。混合震荡，在滚子的地方，和孵化一夜之间在30 ° C。 </li><li>在第二天早上，1毫升的文化（或低密度文化）转移到微管。收获800克的细胞在室温下5分钟离心。 </li><li>从这里开始，工作在通风橱中，以避免吸入有毒气体β-巯基乙醇。小心取出上清液使用移液器，在冰冷的裂解液100μL（4％[V / V] 5 N氢氧化钠，0.5％[V / V]β-巯基乙醇），重悬细胞沉淀。混合大力涡旋，并在冰上孵育30分钟。 </li><li>在孵化期间，确定100μl裂解缓冲液pH值9-10的滴定所需的HCl体积（6 N）。为实现这一目标，10微管在机架和转移裂解液100μL到他们每个人。添加盐酸（6个N）各卷（1-10μL），以每管和涡旋混合。取一次样，检查的使用酸碱指示剂条溶液的pH值。 </li><li>孵化结束，添加量在上一步中的裂解和涡旋混合（更准确地放置在管方没有提示插入裂解盐酸溶液）确定的盐酸。 </li><li>新增50个3倍样品缓冲液（30％[V / V]甘油，15％[V / V]β-巯基乙醇，37.5％[V / V] 500毫米的Tris - HCl pH值= 6.8，0.15％W / V ]十二烷基硫酸钠[SDS]和几粒溴酚蓝）震荡（如果溶液变为黄色，这意味着，pH值过低，应添加到解决方案和几微升裂解液的裂解和混合变为蓝色）。 </li><li>煮沸5分钟后穿刺针的微管盖了一个洞的加热块上裂解解决方案。 </li><li>微管加热块取出，并让溶液在室温下2分钟冷却。 </li><li>涡裂解解决方案，然后旋转10秒。免疫印迹分析，装载到SDS - PAGE凝胶30μL。 </li></ol>五合模试验检测效应引起的生长抑制表型<ol><li>从一个全新的板块中挑选一个酵母细胞集落（直径1-2毫米）携带表达质粒的利益和接种，在15毫升的聚丙烯管，3毫升2％葡萄糖作为碳源的培养基中合成完整的，没有的氨基酸酸或核苷酸，表达质粒提供prototrophy。重复相同的过程控制酵母携带空质粒的应变。文化管放置在滚筒和孵育30 ° C时不断旋转的隔夜。 </li><li>在接下来的一天，从滚筒中删除的文化和离心机在800克，在室温下5分钟。取出离心管上清液。 </li><li>重悬细胞沉淀在3毫升无菌DDW和涡旋混合。 </li><li>重复离心步骤3毫升无菌DDW和悬浮细胞。 </li><li>要确定他们的光密度（OD），每一种文化转移100μL含900微升DDW和旋涡到微管。倒入一个塑料比色皿管的内容和措施，在600 nm波长的吸光度（作为参考，使用1毫升DDW填补了比色皿）。试管文化OD 600乘以10的稀释倍数计算外径 600的初始文化。 </li><li>其次，根据初步培养OD 600，准备用OD 600 = 1.0在无菌微管的细胞悬浮液（1毫升）。 </li><li>通过使用900μL无菌DDW填充的3无菌微管，准备三个10倍连续稀释（ 外径 600 = 0.1，0.01和0.001），从每个细胞悬液（外径600 = 1.0）在上一步准备。 </li><li>准备没有的氨基酸或核苷酸质粒的选择提供prototrophy含有合成完全培养基的两个板块。一个板块的中期应补充2％葡萄糖和半乳糖2％和1％的棉子糖等板块。干在室温下在无菌层流20分钟罩板和他们放在一个网格。 </li><li>对于每一个文化，现货10的抑制和诱导媒体在连续四个稀释液。 </li><li>察觉后，离开几分钟的引擎盖打开板。斑点，干燥，覆盖板，放置在30℃孵化器为2-3天。 </li><li>孵育后，取出板和分析酵母生长。首先，确认细胞密度包含压制介质板类似。然后，比较文化的发展，表达效应蛋白与含有诱导培养基板上的一个空载体的文化的兴趣。 注：效应可能的目标，没有限速标准实验室生长条件下生长酵母细胞的过程。要允许的生长抑制表型鉴定等效应，诱导媒体与酵母细胞功能的改变的化合物，可以补充，从而增加其灵敏度效应。此过程中，都不可能将综合性化学品的清单，请参阅Parsons等。 （2004）2。 </li></ol>六。代表性的成果代表酵母发现III型效应的表达诱导的生长抑制表型的检测和检测如图所示。 1。 HopAA1 - 1和革兰氏阴性植物病原细菌假单胞菌丁香光伏番茄（PST）AvrE1 III型效应AvrPto，在这个实验中，表达着丝粒中含有的酵母菌株BY4741质粒pGML10，并测试他们的能力以抑制酵母菌的生长。半乳糖诱导的GAL1启动子控制下表达PST III型效应，或包含一个空载体的个体酵母文化系列稀释，并镀上压制（葡萄糖）或诱导（半乳糖）媒体（图1）。在镇压介质，进行AvrPto和HopAA1 - 1的表达质粒的酵母菌株展出包含一个空向量的应变作为控制类似的增长。但在同一介质中，酵母携带AvrE1显示的增长略有减少，可能与某种程度渗漏的GAL1启动和AvrE1高的细胞毒作用。在诱导条件下，表达AvrE1效应引起的急剧增长抑制表型，反映在缺乏任何稀释的殖民地。 Munkvold等。3，HopAA1 - 1的表达也导致严重的抑制经济 ​​增长，正如以前观察到的的，而AvrPto没有显示出任何效果。 <img src="/files/ftp_upload/1865/1865fig1.jpg" alt="图1" /> 图1。造成假单胞菌丁香光伏番茄表达的酵母生长的抑制作用（PST）III型效应AvrE1和HopAA1 - 1。酵母菌株（BY4741）的质粒pGML10，要么为空，或携带推动GAL1 -半乳糖诱导表达AvrPto录音带HopAA1 - 1或AvrE1，过夜合成完整的培养基（2％）与葡萄糖作为碳源，而且缺乏亮氨酸。文化分别洗净，标准化外径600发现到合成完整的固体媒体缺乏亮氨酸和含葡萄糖（2％），或半乳糖（2％）和棉子糖（1％）= 1.0，连续10倍稀释。照片后增长2和第3天在30 ° C分别增长酵母中的葡萄糖和半乳糖媒体，采取。

<ol>
	<li>Siggers, K. A., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+Saccharomyces+cerevisiae:+a+versatile+model+system+for+the+identification+and+characterization+of+bacterial+virulence+proteins.">The yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.</a> Cell Host Microbe. 4, 8-15 (2008).</li><li>Parsons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat. Biotechnol. 22, 62-69 (2004).</li><li>Munkvold, K. R., Martin, M. E., Bronstein, P. A., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+Pseudomonas+syringae+pv.+tomato+DC3000+type+III+secretion+system+effector+repertoire+reveals+several+effectors+that+are+deleterious+when+expressed+in+Saccharomyces+cerevisiae.">A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.</a> Mol. Plant-Microbe Interact. 21, 490-502 (2008).</li><li>Curak, J., Rohde, J., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+as+a+tool+to+study+bacterial+effectors.">Yeast as a tool to study bacterial effectors.</a> Curr. Opin. Microbiol. 12, 18-23 (2009).</li><li>Slagowski, N. L., Kramer, R. W., Morrison, M. F., LaBaer, J., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+yeast+screen+to+identify+pathogenic+bacterial+proteins.">A functional genomic yeast screen to identify pathogenic bacterial proteins.</a> PLoS Pathog. 4, e9-e9 (2008).</li><li>Huang, J., Lesser, C. F., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+role+of+the+CopN+protein+in+Chlamydia+pneumoniae+intracellular+growth.">The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth.</a> Nature. 456, 112-115 (2008).</li></ol>

在此演示中，我们演示了如何使用芽殖酵母酿酒酵母作为III型细菌效应蛋白和如何识别效应诱导的生长抑制表型的表达异源系统。重要的是，这些表型可以利用遗传筛选，以确定的负面影响酵母生长的效应抑制器。抑制器可能代表无论是直接的效应研究的目标或蛋白质参与细胞过程的影响的效应。无数III型效应，从动物和植物细菌病原体在酵母中迄今已表示，生长抑制引起的表型，其中几?...

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		<tbody>
		
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<td>Yeast extract</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>212750</td>
<td>&nbsp;</td>
<tr>
<td>Peptone</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>211677</td>
<td>&nbsp;</td>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>214010</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8045</td>
<td>&nbsp;</td>
<tr>
<td>Yeast nitrogen base w/o amino acids</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>291940</td>
<td>&nbsp;</td>
<tr>
<td>Yeast synthetic drop-out medium supplement</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Y2001</td>
<td>&nbsp;</td>
<tr>
<td>D-galactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G0750</td>
<td>&gt;99%; &lt;0.1% glucose</td>
</tr>
<tr>
<td>D-raffinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R0250</td>
<td>&gt;98%</td>
</tr>
<tr>
<td>L-leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
<tr>
<td>Uracil</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>U0750</td>
<td>&nbsp;</td>
<tr>
<td>L-tryptophan</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0254</td>
<td>&nbsp;</td>
<tr>
<td>L-histidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6034</td>
<td>&nbsp;</td>
<tr>
<td>DNA, single stranded, from salmon testes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D7656</td>
<td>&nbsp;</td>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5879</td>
<td>Desiccate</td>
</tr>
<tr>
<td>Hydrochloric acid (HCl)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol (PEG) 3350</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4338</td>
<td>&nbsp;</td>
<tr>
<td>Lithium acetate (LiAc)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4958</td>
<td>&nbsp;</td>
<tr>
<td>Tris (base)</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>4109-02</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediamine-tetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5516</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B6131</td>
<td>&nbsp;</td>
<tr>
<td>Dodecyl sulfate sodium salt (SDS)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>8.22050.1000</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tubes (15 ml)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Sterile</td>
</tr>
<tr>
<td>Spectrophotometer cuvette (10x4x45 mm)</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>67.742</td>
<td>&nbsp;</td>
<tr>
<td>Inoculation loop</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Z643009</td>
<td>Sterile</td>
</tr>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7543</td>
<td>&nbsp;</td>
<tr>
<td>pH indicator strip, pH 6.5-10.0</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1.09543.0001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

identification of growth inhibition phenotypes induced by expression of bacterial type iii effectors in yeast

许多革兰氏阴性致病菌使用III型分泌系统转运到宿主细胞的细胞质效应蛋白套件。在细胞内，III型效应颠覆宿主细胞的过程，抑制免疫反应，促进病原体的生长。无数III型效应的植物和动物的细菌病原体已经确定的日期，但其中只有少数是很好的特点。了解这些效应的功能已经削弱了功能冗余的结合，在一个给定的菌株效应剧目，潜移默化的影响，他们可能对增加的毒性，可能是​​特定于某些感染阶段的角色，并在基因困难操纵某些病原体。 Ⅲ型效应在芽殖酵母中的表达<em&gt;酿酒酵母</em&gt;可能允许绕过这些限制，并援助的效应蛋白的功能特性。由于Ⅲ型效应往往与目标之间酵母和其他真核生物中保守的细胞过程，及其在酵母中的表达可能会导致生长抑制表型，可以被利用来阐明效应的功能和目标。使用酵母细菌效应的功能研究的其它优点包括其遗传的易处理，对他们的ORFs绝大多数预测功能的信息，以及全基因组和小规模实验的众多工具和资源的可用性。在这里，我们讨论设计的酵母细菌Ⅲ型效应蛋白的表达系统的关键因素。这些措施包括适当的启动子驾驶的效应基因（S）的利益的表达，效应基因的拷贝数中，表面抗原的标记，用来验证蛋白的表达，和酵母菌株。我们目前的程序诱导效应在酵母中的表达和免疫印迹验证其表达。此外，我们描述了一个效应诱导的生长抑制表型鉴定琼脂平板上的斑点检测。使用此协议的，可以延长到宿主细胞提供任何病原体和易位机制的致病因素的研究。

Watch this Scientific Journal Video about Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast at JoVE.com

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

在这个视频中，我们描述酵母效应引起的生长抑制表型鉴定的细菌Ⅲ型效应的表达程序。这种表型，随后可利用澄清效应功能和目标。

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

identification-growth-inhibition-phenotypes-induced-expression

Research

JoVE Journal

Biology

16.6K 次观看.  Tel Aviv University. 在这个视频中，我们描述酵母效应引起的生长抑制表型鉴定的细菌Ⅲ型效应的表达程序。这种表型，随后可利用澄清效应功能和目标。

视频: Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...