Signal-amplifying genetic enables in vivo observation circuit of weak promoter activation in the RhI quorum sensing system
Signal-amplifying genetic enables in vivo observation circuit of weak promoter activation in the RhI quorum sensing system
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DOI：
10.1002/bit.20371
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发表时间：
2005-03-20
影响因子：
3.8
通讯作者：
Weiss, R
中科院分区：
文献类型：
--
作者：
Karig, D;Weiss, R
转录活性的微小变化通常会显着影响表型，但通过常规方法在体内无法检测到。为了解决这个问题，我们提出了一种使用信号放大遗传电路来检测弱转录反应的技术。我们应用这种技术来揭示铜绿假单胞菌中几个 RhI 群体感应控制 (qsc) 启动子的先前无法检测到的对数期响应。在大肠杆菌中构建并测试了具有 RhI 启动子和转录放大组件的遗传电路。这使我们能够将所研究的启动子的行为与 Las 和喹诺酮相互作用分开。为了放大 qsc 启动子对酰基高丝氨酸内酯 (AHL) 的反应，将高效 lambda 阻遏基因置于几个 RhI 启动子的下游，并与 lambda P-(R) 启动子控制下的荧光报告基因偶联。通过扩增，在 AHL 诱导和非诱导的培养物之间观察到启动子的荧光水平高达 100 倍的差异，否则这些启动子的反应是无法检测到的。此外，结合使用信号放大和在大肠杆菌中进行实验简化了 AHL 信号串扰的分析。例如，我们发现，在我们的系统中，C4HSL/RhIR 复合物可激活 qscrhIA 和 qscphzA1，而 30C12HSL/RhIR 复合物可激活 qscphzA1，但不会激活 qscrhIA。这种串扰信息特别重要，因为扩增构建体的潜在用途之一是检测环境和临床分离株中的特定群体感应信号。此外，将网络分解为基本部分、在明确的背景中隔离这些组件以及使用放大来表征串扰和同源信号响应的过程体现了理解复杂遗传网络的重要方法。 (C) 2004 年 Wiley 期刊公司。
Small changes in transcriptional activity often significantly affect phenotype but are not detectable in vivo by conventional means. To address this problem, we present a technique for detecting weak transcriptional responses using signal-amplifying genetic: circuits. We apply this technique to reveal previously undetectable log phase responses of several RhI quorum sensing controlled (qsc) promoters from Pseudomonas aeruginosa. Genetic circuits with RhI promoters and transcriptional amplification components were built and tested in Escherichia coli. This enabled us to isolate the behavior of the promoters under study from Las and quinolone interactions. To amplify qsc promoter responses to acyl-homoserine lactones (AHL), the highly efficient lambda repressor gene was placed downstream of several RhI promoters and coupled to a fluorescent reporter under the control of the lambda P-(R) promoter. With amplification, up to similar to 100-fold differences in fluorescence levels between AHL induced and noninduced cultures were observed for promoters whose responses were otherwise not detectable. In addition, the combination of using signal amplification and performing experiments in E. coli simplified the analysis of AHL signal crosstalk. For example, we discovered that while a C4HSL/RhIR complex activates both qscrhIA and qscphzA1, a 30C12HSL/RhIR complex activates qscphzA1 but not qscrhIA in our system. This crosstalk information is particularly important since one of the potential uses of amplification constructs is for the detection of specific quorum sensing signals in environmental and clinical isolates. Furthermore, the process of decomposing networks into basic parts, isolating these components in a well-defined background, and using amplification to characterize both crosstalk and cognate signal responses embodies an important approach to understanding complex genetic networks. (C) 2004 Wiley Periodicals, Inc.