由Springer和爱思唯尔的许可转载的数字大大确认。

1.隔离的E.大肠杆菌共转化<ol><li>准备适当的大肠杆菌的电-感受态细胞大肠杆菌菌株进行改造。转移到1ml LB培养基（胰蛋白胨，酵母提取物，氯化钠10，第5和10克/升，分别地）选择的菌株的单菌落，并在37℃下孵育180 rpm振摇条件下进行。稀释预培养1:500中的新鲜LB培养基25毫升孵育的培养在37℃。 </li><li>在对数相（0.6 OD）离心所述细胞悬浮液在5000×g离心20分钟），并悬浮颗粒中的10％，体积/体积的冰冷无菌甘油 - 水在一半原培养体积。重复此步骤4次，每次再悬浮体积减半。最后，重新悬浮在甘油/水的颗粒和除以细胞悬浮于50μl等分试样。存储等分在-80℃下长达6个月。 </li><li>溶解补充有0.5mM EDTA的在无菌水中的所需质粒。解冻的ICê电感受态细胞的等分试样并混合以载体的合适量（2.5-5纳克）。分配所述混合物成适合于电穿孔0.1 1cm比色杯中，并应用​​1.8千伏。 </li><li>紧接在电穿孔的细胞转移到1毫升SOC培养基（LB培养基补充有0.2％w / v的葡萄糖，10mM的MgCl 2的，2.5毫米氯化钾），孵育1小时下振荡，最后转移100μl的等分试样到培养皿含有LB琼脂用适当的抗生素。孵育O / N在37℃。 </li><li>通过在培养皿裸奔单菌落净化转化。 </li><li>准备主转化电感受态细胞，并重复步骤1.1至1.5进一步质粒进行改造。 </li><li>制备的共转化体的甘油原种。转移在LB单菌落补充有合适的抗生素，孵育在37℃下振荡，并且在对数期（OD 0.6）离心所述细胞悬浮液在5000×g离心20分钟。 řesuspend在LB-抗生素培养基含有15％体积/体积甘油沉淀。在分装分装并储存在 - 80℃。 </li></ol> 2.共表达大肠杆菌的α和ε亚基大肠杆菌 DNA聚合酶III <ol><li>用无菌环传送少量的相应应变甘油（ 例如，TOP10 / PGOOD-ε243和TOP10 / PGOOD-ε243-θ/pBADα1160，参见图1）到含有LB培养基和抗生素的培养皿。让细胞悬浮液干燥到培养皿，并且条纹细胞液滴。在37°CO / N。 </li><li>转让，使用无菌牙签，单菌落到1ml的LB-抗生素培养基中。孵育8小时，在37℃。稀释预培养1:500在新鲜的培养基中孵育在30℃下15小时。 </li><li>添加IPTG，阿拉伯糖，或阿拉伯糖和IPTG，每1毫米。孵育在30℃下2.5小时。收集细胞，并存储该粒料在-20℃。 </li><li>解冻粒料在冰上，重悬在裂解缓冲液（50mM的Tris-HCl，50 mM氯化钠，1mM EDTA中，2.5毫摩尔二硫苏糖醇，1mM苯甲基磺酰氟（PMSF），pH 8）中。轻轻的均匀的细胞悬液与冷玻璃窑匠。 </li><li>声处理，在15瓦下15秒，接着15秒冷却时间间隔（4个循环）。 </li><li>离心的总蛋白提取物在10,000×g离心20分钟，在4℃以回收可溶组分。 </li><li>通过SDS-PAGE（12.5％丙烯酰胺）的每个可溶性蛋白质提取物的等分试样进行分析。为此目的，传输20微升每种可溶性蛋白提取物含有60微升的H 2 O和20微升加样缓冲液（250mM的Tris-盐酸的pH Eppendorf管6.8，10mM的β巯基乙醇，10％（重量/体积） SDS，50％（体积/体积）甘油，0.25％（重量/体积）溴酚蓝），并煮沸5分钟。负载18微升各样品并进行电泳，在140 V为​​约1.5小时。 </li></ol> 3.共表达Deinococ的α亚基对焦球菌DNA聚合酶III和εE.亚基大肠杆菌 DNA聚合酶III <ol><li>制备E的预培养大肠杆菌菌株TOP10 /的pBAD-α 博士 / PGOOD-ε243在1ml LB-氨苄青霉素-四环素的培养基中孵育8小时，在37℃。稀释1:1000的新鲜培养基和培养O / N在37℃。 </li><li>稀释1:100的入200ml新鲜的培养基，孵育在37℃下进行3小时，然后诱导3小时与阿拉伯糖和IPTG 1 mM的每种。收获细胞，并将沉淀储存于-20℃。 </li><li>重悬沉淀在50mM的Tris-HCl pH为8，150 mM氯化钠，1mM EDTA中，1毫PMSF。均质并如2.4和2.5中所述破碎细胞。 </li><li>立即离心细胞裂解物以10,000×g离心20分钟。弃沉淀。过滤用布氏漏斗装有3层纸过滤上清液。适用于温和的真空。为了避免过多的泡沫，保持真空锥形瓶冰过滤过程中。 </li><li>根据布拉德福德14确定蛋白浓度。 </li></ol> 4.凝胶过滤层析<ol><li>平衡水夹套16x70（200）用50mM的Tris-HCl，150mM的氯化钠，1mM EDTA中，pH值8.加载至色谱柱上的可溶性蛋白提取物的凝胶过滤柱。为了获得最佳的分辨率，使用1毫升样品环。 0.6毫升/分钟下进行层析。保持柱温在4℃贯穿。 </li><li>收集0.9毫升馏分并通过SDS-PAGE分析它们。转移20微升的每个相关部分，以含有60微升的H 2 O和20微升加样缓冲液（250mM的Tris-盐酸pH值为6.8，10mM的β巯基乙醇，10％（重量/体积）SDS，50％的Eppendorf管中（体积/体积）甘油，0.25％（重量/体积）溴酚蓝），并煮沸5分钟。负载18微升各样品并进行电泳，在140 V为​​约1.5小时。 </li><li>确定的3&#39;-5&#39;核酸外切酶和每个级分在96我们的DNA聚合酶活性LL微孔板。使用胸苷-5&#39;-单磷酸对硝基苯酯（ 对 NP-TMP）作为基板15测定的核酸外切酶的活性。估计使用PPX（焦磷酸酶，嘌呤核苷磷酸化酶，黄嘌呤氧化酶）酶偶联测定法16和DNA聚合酶活性的2-（4-碘苯基）-3-（4-硝基苯基）-5-苯基-2H-四唑鎓氯化物（INT）的作为电子受体16。 </li></ol>人群共同表达E.的野生型ε亚基5.突变分析大肠杆菌 DNA聚合酶III和诱变εD12A变<ol><li>转移大肠杆菌的单个菌落大肠杆菌 TOP10含有的pBAD-ε和PGOOD1-εD12A11矢量到1ml的LB-抗生素培养基中。孵育O / N在37℃。 </li><li>稀释预培养1:250在3瓶中含有新鲜培养基（10毫升）中，添加相应的诱导剂（阿拉伯糖，IPTG，或阿拉伯糖和IPTG 1 mM的每种）孵育在37℃下8小时。制备平行非诱导培养物。 </li><li>收集1毫升的等分试样，然后稀释1:在含新鲜培养基（10毫升）中的新培养瓶中500补充或不使用适当的诱导剂。孵育O / N在37℃。 </li><li>重复步骤5.2和5.3，并收集等分1毫升。 </li><li>确定发生在各培养世代的数目。转移在LB板，100微升的接种和培养合适的连续稀释。孵育O / N在37℃。算在LB平板上的菌落，并计算细胞中存在的接种物（日志I）和在培养在生长（日志C）的端部的数目的对数。要确定代的数量，使用的公式:（日志ç -登录I）/0.301。 </li><li>离心机在5000×g离心20分钟，重悬细胞于1ml的50mM的Tris-HCl pH 7.6的50 mM氯化钠。通透细胞，持续20秒添加2-3滴氯仿并涡旋。 </li><li>确定β-在一个96孔微量每个等分试样的葡糖苷酶活性，使用对 -硝基苯基β-D-吡喃葡糖苷（PNPGluc）作为底物。添加到每孔100μl透化细胞，并加入100μl底物（16毫克/毫升的储备溶液中的H 2 O）。请注意，以避免气泡孔中。读取吸光度在420nm处，用酶标仪及相应的过滤器。 </li></ol>

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W., Ponticelli, S. K. S., Duzen, J. M., Sutton, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+Escherichia+coli+DNA+polymerase+to+the+&#946;-sliding+clamp.">Differential binding of Escherichia coli DNA polymerase to the &#946;-sliding clamp.</a> Mol. Microbiol. 65 (3), 811-827 (2007).</li><li>Greener, A., Callahan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XL1-red:+highly+efficient+random+mutagenesis+strain.">XL1-red: highly efficient random mutagenesis strain.</a> Strategies. 7, 32-34 (1994).</li><li>Chanda, P. K., Edris, W. A., Kennedy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+set+of+ligation-independent+expression+vectors+for+co-expression+of+proteins+in+Escherichia+coli.">A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.</a> Protein Expr. Purif. 47 (1), 217-224 (2006).</li><li>Sun, J., Hopkins, R. C., Jenney, F. E., McTernan, P. M., Adams, M. W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+and+maturation+of+an+NADP-dependent+[NiFe]-hydrogenase:+a+key+enzyme+in+biofuel+production.">Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.</a> PloS One. 5 (5), (2010).</li></ol>

The authors declare that they have no competing financial interests.

蛋白质可以是内在无序，设有区域的三级结构不局限于构25的数量有限。这些无序的蛋白质通常容易发生聚集25，以及它们的分离和表征可能代表一个困难的任务。 E.的ε亚基大肠杆菌 DNA聚合酶III具有两个不同的结构域26,27，即:i）第N-TER域，轴承的3&#39;-5&#39;外切酶活性，并且在主管结合θ亚基; ⅱ）在C-之三结构域，负责结合到α（聚合酶）亚基。 ε的C之三结?...

表达技术的引入提高了低拷贝数酶或者生化研究和工业生产的药理学活性的蛋白质（ 如胰岛素）的。因为这些技术的出现，显著的进步已经实现，以增加重组蛋白的产量和质量。此外，原核1,2和真核3过表达系统被开发，多年来，将提供到蛋白质生物技术， 即，大肠杆菌的"工作马"有用的替代品。替代的平台，以大肠杆菌 ，特别是可获得大肠杆菌导致生产的重组肽或蛋白轴承翻译后修饰的。然而，应该指出的是，E。大肠杆菌仍然代表所选择的生物体为重组蛋白的生产。这是由于多种因素，其中最相关的可认为:I）的不少表达系统（表达载体和应变的可用性）的E.大肠杆菌 1,2; ⅱ）短的产生时间，和高生物质产量，E的大肠杆菌中的各种丰富的和合成媒体; iii）所述浅显操纵或者在生物化学，并在该微生物的基因水平; ⅳ）能产生毒性蛋白4的菌株的分离; V）株具有均匀的感应在群体水平上5,6建设。另外，最近显示适合于生产在大肠杆菌中的表达系统翻译后修饰的蛋白质的大肠杆菌可以设计和构造2。 目前，蛋白质表达主要用来获得单体或同型寡聚蛋白质，其hypersynthesis可以与单个基因克隆到合适的质粒来进行。然而，关注的是最近支付给大肠杆菌的构建大肠杆菌蛋白的共表达系统，具有挑战性的生产， 在体内的杂寡聚复合物2。有趣的是，蛋白质共同表达的早期实验解决了蓝藻核酮糖-1,5-二磷酸羧化酶/加氧7,8的大，小亚基的种间组装，和HIV-1的截断和全长形式的关联逆转录9。这些开创性的研究表明，蛋白质的共表达代表了强大的替代传统的体外重组。此外，蛋白的共表达在大肠杆菌中的大肠杆菌是用于生产不同的蛋白质轴承的翻译后修饰2，得到含非天然氨基酸的蛋白质2，并增加过表达的膜蛋白2的产率。而且，蛋白的共表达的作为工具的电位以赋予大肠杆菌大肠杆菌竞争NCE蛋白质分泌正在积极调查2。 蛋白的共表达在大肠杆菌中的两种主要的策略大肠杆菌可以追求是:i）使用单个质粒来承载不同的基因的过表达; ⅱ）在单细胞中使用多个质粒共同表达靶蛋白。在第一种情况下，该标准的质粒的选择不与那些传统的单一蛋白质表达实验不同，虽然含有串联启动子/操纵元件特定的质粒用于共表达10构成。因此，这第一种方法是相当简单的。然而，应当提到的是，使用一个单一的质粒来共表达不同蛋白质面临两个主要的困难:i）的矢量随承载基因的数量，限制共表达蛋白的数量的分子质量; ⅱ）当多个基因的单个启动子的控制下克隆，极性可以增减E从启动子远端的基因的表达。单E.采用双重或多重质粒大肠杆菌细胞具有完成所选择的载体的相容性，因此，施加约束以质粒的合格组合。然而，该第二共表达策略设有含载体的分子质量的优点，并限制极性。我们最近建造设计，方便的共表达质粒11之间的基因的穿梭蛋白共表达系统。特别是，我们构建了PGOOD矢量系列，相关特征，其中是:i）复制的一个P15A原点，以提供PGOOD质粒与含有市售载体的ColE1原点（ 例如，所述的pBAD系列12）的相容性; ⅱ）四环素抗性盒; III）LAC存在派生调控元件， 即启动操作员（O 1）夫妇和的lacI </eM&gt; Q基因。使用适当的pBAD-PGOOD夫妇，我们能够以过表达大肠杆菌的催化核心大肠杆菌 DNA聚合酶III，三种不同的亚单位组成， 即，α（5&#39;-3&#39;聚合酶），ε（3&#39;-5&#39;外切核酸酶）和θ（稳定ε）13。特别是，我们表明，共表达αεθ复杂的是严格依赖于除大肠杆菌两者的IPTG和阿拉伯糖的大肠杆菌培养液，从PGOOD和的pBAD，分别为（ 图1A）触发过表达。 在本报告中，我们说明了如何蛋白共同表达可以有效地解决与蛋白复合物亚基的溶解性差的困难。此外，我们显示了如何在体内蛋白互补试验可以进行的，我们终于在大肠杆菌中使用的蛋白质共表达来调节突变频率报告关口我。为了这个目标，我们使用适合来说明每个案例研究相关的例子PGOOD-的pBAD夫妇。

<table border="0" cellpadding="0" cellspacing="0" width="627">
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			<td style="width:119px;">70161</td>
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			<td height="21" style="height:21px;width:216px;">Acrylamide solution 30%</td>
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			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0719</td>
			<td>For gel electrophoresis</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cuvettes 0.1 cm</td>
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			<td style="width:119px;">1652089</td>
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	</tbody>
</table>

the multifaceted benefits of protein co-expression in escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

E.的ε亚基大肠杆菌 DNA聚合酶III由243个氨基酸，并设有溶解性差17,18，除非残留187-243删除17。然而，我们以前曾表明11的共表达的全长α，ε和θ亚基产生可溶的DNA聚合酶III催化核心（ 图1）。特别是，使用的pBAD-PGOOD共表达系统，我们证明:ⅰ）α和ε亚基的过表达能够独立地通过阿拉伯糖和IPTG，分别为（ 图1A）进 ​​行控制; II）全长ε?...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K 次观看.  University of Bologna. Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells. 

视频: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

科研

生物学

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157:H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.