A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter
A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter
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DOI：
10.1016/j.ab.2004.03.064
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发表时间：
2004-07-01
影响因子：
2.9
通讯作者：
Karp, M
中科院分区：
文献类型：
--
作者：
Pellinen, T;Huovinen, T;Karp, M
一种无细胞生物传感器，用于检测特定的小分子配体的转录诱导。作为模型系统，使用四环素和汞诱导的启动子含有萤火虫荧光素酶作为报告基因。制备大肠杆菌S30提取物并用于偶联转录-翻译反应。通过使用纯化的泰特阻遏蛋白和MerR调节蛋白，我们可以研究阻遏-操纵子相互作用，以优化每个组件的相对浓度。以前，四环素和汞的检测使用类似的转录调控在整个活细胞中进行。与全细胞生物传感器相比，我们的研究结果表明，更好的灵敏度检测四环素和汞的毒性作用，避免了在无细胞系统。此外，由于该系统省略了细胞培养和细菌膜作为分子通道抑制剂，因此可以在短得多的时间内进行测定，而不使用遗传修饰的生物体。(C)2004年爱思唯尔公司All rights reserved.
A cell-free biosensor for the detection of transcription induction by specific small-molecule ligands is presented. As model systems, tetracycline and mercury-inducible promoters were used containing firefly luciferase as reporter gene. Escherichia coli S30 extract was prepared and used for coupled transcription-translation reactions. By using purified Tet repressor and MerR regulatory proteins, we could study repressor-operator interactions for optimizing the relative concentrations of each component. Previously, detection of tetracycline and mercury using similar transcriptional regulation in whole living cells has been carried out. As compared to whole-cell biosensors, our results showed better sensitivity for the detection of tetracycline and the toxic effect of mercury was avoided in the cell-free system. Also, as the system omits cell cultivation and bacterial membranes as molecule passage inhibitors, it is possible to carry Out assays in much shorter times and without the use of genetically modified organisms. (C) 2004 Elsevier Inc. All rights reserved.