Name: identification of growth inhibition phenotypes induced by expression of bacterial type iii effectors in yeast
Uploaded: 2010-03-30T16:30:00
Description: Watch this Scientific Journal Video about Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast at JoVE.com

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...

<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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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
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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 ...

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