我们感谢美国国土安全部美国提供资金的一部分。

1。细菌菌株和DNA模板制备<ol><li>成长的大肠杆菌菌株大肠杆菌 O157：H7，非O157和其他致病菌种，如沙门氏菌和志贺氏菌，在37℃过夜振荡。 </li><li>使用适当的工具包（见表材料），并按照制造商的说明书提取细菌培养物的DNA。 </li><li>测量光密度（OD 260）通过使用分光光度计来确定DNA的浓度。 </li></ol> 2。引物和探针设计的qPCR实验方法<ol><li> 大肠杆菌 O157：H7的序列从GenBank登录号AE00517411。设计引物和探针使用适当的软件进行引物和探针设计，实时荧光定量PCR（见材料明细表）。 是引物和探针的序列如下：Z3276-正向（F）5&#39;-TATTCCGCGATGCTTGTTTTT-3&#39; Z3276-反向（R）5&#39;-ATTATCTCACCAGCAAACTGGCGG-3&#39; Z3276-Probe公司，FAM-CCGCAATCTTTCC-MGBNFQ </li><li>重组的引物粉末与水，导致在10μM作为引物工作溶液的浓度。 </li><li>稀释探头到10微米（μm）探头的工作方案。标记探针的效力与批次不同而不同。因此，使用以获得最佳性能的qPCR滴定前每批标记的探针。 </li></ol> 3。设置定量PCR检测条件<ol><li>制备反应混合物，它由25.0微升2倍的实时PCR主混合物，200nM的正向引物，200nM的反向引物，探针和100纳米。 </li><li>添加5微升的样本DNA（100皮克）和水的适当体积达到50微升的最终体积。 </li><li>对于非模板控制，使用5微升的水来代替DNA样本。 </li><li>设置定量PCR条件如下：95℃保持10分钟进行活化的TaqMan的;接着40个循环的95℃10秒变性和60及＃176，C为1分钟的退火/延伸。 </li></ol> 4。定量PCR灵敏度测试<ol><li>使从中期指数期的培养一个连续10倍稀释液（OD 600 = 0.5，约1.5×10 8 CFU / ml的通过电镀）。 </li><li>加入100 ml细胞稀释到96孔板中一式三份。 </li><li>离心板上，在2500×g离心10分钟。 </li><li>加入50毫升的DNA提取液到每个孔中。 </li><li>重悬细胞沉淀用多通道移液器。 </li><li>用薄膜密封板，熬板在水浴10分钟，离心在2500×g离心2分钟。 </li></ol> 5。准备活的和死细胞混合物的PMA处理<ol><li>成长10毫升E的大肠杆菌 O157：H7在37°C至指数中期阶段，并划分成文化两等份。 </li><li>在水浴中死细胞煮沸细胞的一个等份10分钟，并保持其他等分试样为活细胞。 </li><li>验证再从热​​灭活等分无活细胞通过电镀在LB琼脂平板上的细胞。 </li><li>取2毫升活和热灭活的细胞，并调整浓度至8×10 6 CFU /毫升的LB培养基中。创建四套细胞稀释液从8×10 0〜8×10 6 CFU /毫升。 </li><li>使用前两组细胞稀释液与PMA或未经处理的活细胞。 </li><li>到第三和第四组的细胞稀释液，加入8×10 6个死细胞，以每细胞稀释液（8×10 0 - 8×10 6），以使活的和死细胞的混合物。 </li><li>对待第三套稀释的PMA和使用第四套稀释控制。 </li></ol> 6。处理细胞用PMA <ol><li>溶解PMA在二甲亚砜（或水），使10mM储备溶液并储存在-20℃下在黑暗中。 </li><li>分装400微升的活细胞，死细胞和活细胞和死细胞的混合物三个独立的微管。 </li><li>加入2.0毫升10毫PMA对每个等分试样的细胞，以50μM的终浓度。 </li><li>孵育样品在环境温度下5分钟在黑暗中，轻轻摇动几次，3秒每次。 </li><li>加入100微升的样品在96孔板中。 </li><li>密封板的光学膜，并把该板在冰上。 </li><li>从650瓦卤素光源20 cm套板和暴露2分钟的曝光。 </li><li>离心板上，在2500×g离心10分钟，轻轻弃去上清液，并仔细漏板的一块吸收纸。 </li><li>在50μlDNA提取溶液重悬细胞沉淀用移液器上下吹吸20次，一个多通道的Pipetman。 </li><li>用薄膜密封板，煮沸水浴10分钟，离心2,500 xg离心2分钟。在板的上清液是从用PMA和准备的qPCR处理的细胞的DNA中。 </li><li>执行定量PCR作为记述的床的上方，用5微升的DNA。 </li></ol>

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作者什么都没有透露。

该研究的主要目的包括使用的ORF Z3276，独特的为E。大肠杆菌 O157：H7，作为定量PCR检测一个唯一的生物标志物。目前，大多数qPCR实验的目标致病基因，如STX1，STX2和善良 1,2,5,6或共享表型的基因，如uidA基因3,4，rfbE和的fliC基因8,9。偶尔，一个种或菌株不能明确的毒力基因（S）确定的，如1和STX STX 2基因，这些基因存在于不同的种或菌株16为好。使用rfbE和fliC的对靶基因，就需要这两个基因以用于完整的识别17。使用ORF Z3276作为生物标志物，这qPCR分析已被证明是敏感和特异的测定（ 表1）。该测定法已受到严格的包容性和排他性测试，包括O157：H7菌株（n = 3的67），100多个非O157菌株，和其他致病菌种，如沙门氏菌和志贺氏菌 。所有O157：H7检测菌株呈正检测到，并且没有交叉反应性，发现从非O157菌株（ 表一 ），表明ORF Z3276是一种独特的和强大的生物标志物的检测大肠杆菌的大肠杆菌 O157：H7。 该研究的另一个目的是通过PMA处理的DNA提取之前选择性地检测活细胞的死细胞。 PMA可以渗透到死细胞受损的膜和与DNA结合，从而抑制死细胞中PCR扩增13的DNA扩增。我们已经修改对PMA处理的程序，并适应它为过程的96孔板格式。的曝光正确的强度是必不可少的，以获得最佳的交联作用。以前，微管已被用于这个步骤13-15。然而，单独的微管是难以处理在光前曝光的步骤，而这些管子不是绝对透明的，以获得最佳的跨喜好。使用96孔板，我们改进PMA-处理的效率。这些变化可能会影响测定在几个方面：它们可以更容易地获得彻底和均匀的交联效果，特别是与许多样本;处理样品可以从一个板移动到另一个，以使其能够用于检测大量样品的，他们提供的自动化潜力的全过程这PMA-qPCR分析。这PMA-qPCR分析的局限性在于，PMA处理增加了PCR分析的成本有所并且稍微降低PCR检测的灵敏度。

PCR是一种常用的技术进行检测大肠杆菌的大肠杆菌 O157：从食品和环境样本H7。确定为E.特定生物标志物大肠杆菌 O157：H7是其中的关键因素成功的检测PCR方法之一。常用的PCR分析检测大肠杆菌的生物标志物大肠杆菌 O157：H7包括志贺毒素（STX 1和STX 2）1,2，uidA基因 3,4， 善良 5,6，rfbE 7，和的fliC基因8,9。这些生物标志物可以提供可变的效率识别，然而，大多数的生物标志物不能被用作唯一的生物标记物进行检测大肠杆菌的大肠杆菌 O157：H7。这种不足在靶基因的分化功率要求更具体，更强大的生物标志物（s）的鉴定大肠杆菌的大肠杆菌 O157：H7 10。 众多探针用于在大肠杆菌中的基因或假想的开放阅读框（ORFs） 大肠杆菌O157：H7型11被设计为基于本实验室的DNA基因分型芯片，并通过了国家生物技术信息中心的GenBank数据库中寻找线索qPCR分析。 Z3276的ORF，它是一种菌毛基因11的序列被选择为qPCR分析。随后，qPCR分析通过对PCR参数如浓度的引物和探针，以及退火和延伸温度（数据未示出）多次试验的开发。经过对参考菌株，如大肠杆菌激励包容性和排他性测试大肠杆菌 O157：H7，非O157和志贺氏菌在定量PCR中，ORF Z3276被认定为一个特定的和强大的遗传标记用于识别E的大肠杆菌 O157：H7。然后，我们开发使用这种独特的生物标志物的鉴定大肠杆菌的一个新的定量PCR方法大肠杆菌 O157：H7。 在检测大肠杆菌的另一个挑战大肠杆菌 O157：H7的污染FO消耗臭氧层物质和环境的准确测定从样品中的活细胞。 DNA的死亡细胞和不能区分存活在PCR法的延伸存在可能导致假阳性读数，从而导致在检测活细胞数的高估。因此，这限制了在PCR中使用的精确检测食源性致病菌12。一种新颖的方法来区分死细胞的DNA已被采取了在过去的几年里13-15结合单叠氮丙啶（PMA）处理与PCR检测方法。 PMA是能去的死细胞内，并相互插入到DNA，当暴露于光，但不能去活细胞的内部，以与活细胞的DNA反应。因此，在活细胞和死细胞的PMA处理的混合物中，死细胞的DNA不能在PCR扩增13。我们结合PMA治疗新开发的qPCR分析选择性地检测活的大肠杆菌大肠杆菌 O157：H7细胞。此外，我们已经取得了PMA-qPCR的一SSAY可以用于高通量检测。

<table border="0" cellpadding="0" cellspacing="0" width="901">
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	</colgroup>
	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">AB 7900HT Fast Real-time PCR system</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">2 x Universal master mix</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4304437</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PrepMan Ultra</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4318930</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Primer Express</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="58" rowspan="2" style="height:58px;width:249px;">Probes</td>
			<td rowspan="2" style="width:164px;">ABI</td>
			<td>Top of Form</td>
			<td rowspan="2"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">4316033 Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PMA</td>
			<td style="width:164px;">Biodium</td>
			<td align="right" style="width:171px;">40013</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="87" rowspan="3" style="height:87px;width:249px;">Puregen cell and tissue kit</td>
			<td rowspan="3" style="width:164px;">Qiagene</td>
			<td>Top of Form</td>
			<td rowspan="3"></td>
		</tr>
		<tr height="29">
			<td align="right" height="29" style="height:29px;">158622</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Bottom of Form</td>
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		<tr height="29">
			<td height="29" style="height:29px;width:249px;">DU530 spectrophotometer</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Spectrophotometer</td>
			<td style="width:164px;">NanoDrop Technology</td>
			<td style="width:171px;">&#160;ND-1000</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">650 w halogen light source</td>
			<td style="width:164px;">Britek</td>
			<td style="width:171px;">H8061S</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Otptical Adhesive film</td>
			<td>ABI</td>
			<td align="right">4311971</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Optical 96-well reaction plate</td>
			<td>ABI</td>
			<td align="right">4316813</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of live escherichia coli o157:h7 cells by pma-qpcr

独特的开放阅读框（ORF）Z3276被确定为特定遗传标志为E.大肠杆菌 O157：H7。 qPCR实验是检测大肠杆菌的开发大肠杆菌 O157：H7通过靶向ORF Z3276。用此方法，我们可以检测低至大肠杆菌的DNA的基因组的拷贝数大肠杆菌 O157：H7。该测定的灵敏度和特异性通过用大量的大肠杆菌的密集验证试验被证实大肠杆菌 O157：H7的菌株（N = 369）和非0157株（N = 112）。此外，我们结合了单叠氮丙啶（PMA）过程与新开发的qPCR协议，用于选择性检测从死细胞的活细胞。从PMA治疗的死细胞DNA扩增几乎完全相反，从PMA处理的活细胞中的DNA几乎不受放大抑制。此外，该协议已经被修改，并适于为一个简单的，一致的处理大量样品的96孔板格式。 Ŧ他的方法，预计将有准确的微生物和流行病学监测食品安全和环境光源的影响。

检测大肠杆菌的大肠杆菌 O157：使用ORF Z3276 H7通过qPCR 一个这种测定法的关键因素是在定量PCR中使用的Z3276探针的灵敏度和特异性。它可以检测低至大肠杆菌的DNA的基因组的拷贝数大肠杆菌 O157：H7， 如图1A所示 。检查了120个非O157菌株，包括六个主要非O157 STEC菌株O104的：H4菌株， 沙门氏菌 ， 志贺氏菌和菌株，无交叉反应性，发现，如表1所示。 在PMA处理与定量PCR结合 E.孵化大肠杆菌 O157：H7细胞用PMA（50毫米）5分钟在黑暗中进行曝光2分钟被选定为PMA处理条件。此外，我们修改PMA处理程序。与在交联工序中使用的各种透明微管相比，一个96孔板被发现是更有效的达到最佳的交联效果，并比光曝光过程中处理大量的样品管的更方便。 从PMA处理的活细胞和死细胞的定量PCR DNA扩增从这一定量PCR检测死细胞DNA扩增的PMA介导的抑制效应，采用DNA从两个串行10倍活的或死的细胞稀释液中提取出来， 如图1所示。结果表明，从用PMA（红色曲线）和无PMA（蓝色曲线）处理的活细胞产生的曲线似乎线性和几乎彼此相同。轻微的CT值差异，用PMA和没有PMA（ 图1A）处理的活细胞之间所示，表明PMA处理对活细胞中的定量PCR的DNA扩增的影响不大。但一个15 - CT值差异（32,000倍）与PMA处理的死细胞之间，没有物业管理公司，显示演示nstrating的PMA处理有效地抑制DNA扩增的死细胞（ 图1B）。 从定量PCR活的和死细胞混合物检测活细胞两套活细胞稀释液（8×10 0 - 8×10 6 CFU）与​​PMA或不PMA治疗，检测活细胞的活/死细胞混合物。在定量PCR的结果（ 图2A）表明CT值作为相关处理的活细胞（紫色条），以未处理的样品（蓝色条）的数目的并联逆渐进趋势。处理后的活细胞产生了比未处理的活细胞的一个稍高的CT值。在CT值类似的逆渐进趋势观察到活细胞在用PMA（绿色棒）在图2B中被视为良好的活/死细胞的混合物的实数。此外，CT值的这种下降趋势是尽管106死细胞的存在与活细胞中的混合物的数量变化。这些结果表明，该活/死细胞混合物的CT值表示从单独的活细胞中的DNA，而从死细胞中的DNA扩增效率由PMA处理抑制。但是，用PMA治疗的活/死细胞混合物的CT值波动和没有反映在图2B中的（黄色条）的混合物中的活细胞数。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig1highres.jpg" src="/files/ftp_upload/50967/50967fig1.jpg" /> 图1。活细胞和死细胞用PMA或没有在PMA qPCR分析处理（A）的连续10倍稀释E.直播大肠杆菌 O157：H7细胞稀释液（8×10 0 - 8×10 6 CFU）分别用PMA或没有PMA。CT值代表了qPCR实验的一式三份的平均CT值。 （B）用PMA和无PMA在第q比较qPCR分析处理的活细胞和死细胞的DNA扩增的比较。 ΔRn是指荧光强度的变化。 <a href="https://www.jove.com/files/ftp_upload/50967/50967fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig2highres.jpg" src="/files/ftp_upload/50967/50967fig2.jpg" /> 图2。从由PMA-qPCR的四组的细胞培养物的10倍稀释液（8×10 0 - 8×10 6 CFU）的活/死细胞混合物分化的活细胞所指示而进行。 （ 一 ）第一组细胞稀释液用PMA处理，而第二个单元是稀释或未经处理的DNA提取之前。（B）8×10 6个死细胞与所述第三组和第四组的细胞稀释液，使两套活/死细胞的混合物所指示的。一组的细胞混合物中的溶液用PMA治疗，而另一组是DNA提取之前没有PMA处理。每个条形代表一式三份实验±标准差的平均CT值。 <table><tbody><tr><td> 生物 </td><td> 血清型 </td><td> 供试菌株数 </td></tr><tr><td rowspan="15"> 大肠杆菌 </td><td> 2006菠菜O157：H7菌株爆发</td><td> 186 </td></tr><tr><td> 2006 Taco Bell的O157：H7菌株</td><td> 58 </td></tr><tr><td> 2006年约翰·塔克O157：H7菌株</td><td> 11 </td></tr><tr><td>其他0157 DMB应变集合：H7爆发</td><td> 112 </td></tr><tr><td> O104：H4 </td><td> 3 </td></tr><tr><td> O26 </td><td> 18 </td></tr><tr><td> O103 </td><td> 13 </td></tr><tr><td> O111 </td><td> 24 </td></tr><tr><td> O121 </td><td> 2 </td></tr><tr><td> O45 </td><td> 5 </td></tr><tr><td> O145 </td><td> 9 </td></tr><tr><td> O113 </td><td> 2 </td></tr><tr><td> O91 </td><td> 1 </td></tr><tr><td> O55 </td><td> 9 </td></tr><tr><td>其他</td><td> 19 </td></tr><tr><td rowspan="2"> 沙门氏菌 </td><td>伤寒</td><td> 1 </td></tr><tr><td>新港</td><td> 2 </td></tr><tr><td> 痢疾志贺菌 </td><td> 6 </td><td> 2 </td></tr><tr><td> 宋内志贺氏菌 </td><td>未知</td><td> 2 </td></tr><tr><td> 福氏痢疾杆菌 </td><td> 4 </td><td> 1 </td></tr><tr><td> 鲍氏志贺氏菌 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="2">总</td><td> 481 </td></tr></tbody></table> Ŧ能够1。 用于验证实时PCR检测鉴定大肠杆菌菌株大肠杆菌 O157：H7。

Watch this Scientific Journal Video about Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR at JoVE.com

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

H7定位独特的遗传标记，Z3276：一个定量PCR法检测检测大肠杆菌 O157发展。在定量PCR是结合单叠氮丙啶（PMA）处理活细胞检测。这个协议已经被修改，并适于方便和一致地处理大量样品的96孔板格式

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. ...

detection-of-live-escherichia-coli-o157h7-cells-by-pma-qpcr

Research

JoVE Journal

Biology

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

16.5K 次观看.  Food and Drug Administration.  H7定位独特的遗传标记，Z3276：一个定量PCR法检测检测大肠杆菌 O157发展。在定量PCR是结合单叠氮丙啶（PMA）处理活细胞检测。这个协议已经被修改，并适于方便和一致地处理大量样品的96孔板格式

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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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 ...

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 ...

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 ...

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.

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 ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

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.