A synthetic three-color scaffold for monitoring genetic regulation and noise.
A synthetic three-color scaffold for monitoring genetic regulation and noise.
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DOI：
10.1186/1754-1611-4-10
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发表时间：
2010-07-21
影响因子：
5.6
通讯作者：
Elowitz MB
中科院分区：
文献类型：
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作者：
Cox RS 3rd;Dunlop MJ;Elowitz MB
目前用于分析自然调控网络的动态和量化合成电路功能的方法受到缺乏良好表征的遗传测量工具的限制。荧光报告已被用于测量动态基因表达，但最近试图在单细胞中同时监测多个基因没有集中在独立的，孤立的测量。多个报告基因可用于观察天然基因之间的相互作用，或促进生物工程遗传网络的“调试”。在单个细胞中使用三个可区分的报告基因可以揭示仅从一个或两个报告基因无法获得的信息。多个报告子的一个应用是使用遗传噪声来揭示基因之间的调节连接。天然和合成系统中的实验将受益于用于表达多个报告基因和合成网络组件的良好表征的平台。我们描述了这样一个基于质粒的平台，用于合成基因网络的设计和优化，以及内源基因网络的分析。该网络支架由三个可区分的荧光报告基因组成，由诱导型启动子控制，具有方便放置的限制性位点以使修饰简单。我们用单细胞荧光成像和延时显微镜定量表征了大肠杆菌中的支架。这三种光谱不同的报告基因允许独立监测遗传调节和分析遗传噪音。作为一个新的应用程序，这个工具，我们表明，遗传噪音的存在下，可以揭示转录共调节由于一个隐藏的因素，并可以区分组成性调控基因的表达。我们已经构建了一个通用底盘，其中来自天然基因或合成网络组件的三个启动子可以很容易地插入并使用优化的荧光蛋白报告基因在单个构建体上独立监测。我们已经定量表征了底盘的基线行为，以便它可以用于测量动态基因调控和噪声。总的来说，该系统对于分析自然遗传网络和组装合成遗传网络都很有用。
Current methods for analyzing the dynamics of natural regulatory networks, and quantifying synthetic circuit function, are limited by the lack of well-characterized genetic measurement tools. Fluorescent reporters have been used to measure dynamic gene expression, but recent attempts to monitor multiple genes simultaneously in single cells have not focused on independent, isolated measurements. Multiple reporters can be used to observe interactions between natural genes, or to facilitate the 'debugging' of biologically engineered genetic networks. Using three distinguishable reporter genes in a single cell can reveal information not obtainable from only one or two reporters. One application of multiple reporters is the use of genetic noise to reveal regulatory connections between genes. Experiments in both natural and synthetic systems would benefit from a well-characterized platform for expressing multiple reporter genes and synthetic network components. We describe such a plasmid-based platform for the design and optimization of synthetic gene networks, and for analysis of endogenous gene networks. This network scaffold consists of three distinguishable fluorescent reporter genes controlled by inducible promoters, with conveniently placed restriction sites to make modifications straightforward. We quantitatively characterize the scaffold in Escherichia coli with single-cell fluorescence imaging and time-lapse microscopy. The three spectrally distinct reporters allow independent monitoring of genetic regulation and analysis of genetic noise. As a novel application of this tool we show that the presence of genetic noise can reveal transcriptional co-regulation due to a hidden factor, and can distinguish constitutive from regulated gene expression. We have constructed a general chassis where three promoters from natural genes or components of synthetic networks can be easily inserted and independently monitored on a single construct using optimized fluorescent protein reporters. We have quantitatively characterized the baseline behavior of the chassis so that it can be used to measure dynamic gene regulation and noise. Overall, the system will be useful both for analyzing natural genetic networks and assembling synthetic ones.