proTeOn and proTeOff, new protein devices that inducibly activate bacterial gene expression.
proTeOn and proTeOff, new protein devices that inducibly activate bacterial gene expression.
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proTeOn 和 proTeOff 是诱导激活细菌基因表达的新型蛋白质装置。
DOI：
10.1021/cb200168y
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发表时间：
2011
影响因子：
4
通讯作者：
Kaznessis,YiannisN
中科院分区：
文献类型：
--
作者：
Volzing,Katherine;Biliouris,Konstantinos;Kaznessis,YiannisN
使用原始的工作流程，我们已经建模、构建和表征了两个新的分子装置，它们可以诱导激活基因在大肠杆菌中的表达。原核Teton和原核TetOff装置是通过将可诱导的DNA结合蛋白结构域融合到转录激活结构域并构建互补的合成启动子序列来控制下游基因表达而构建的。特别是，反式激活剂是使用四环素抑制物TetR的变体和LuxR激活剂的反式激活域构建的。互补启动子序列包括TetR‘s操作符Teto和LUX启动子元件。这些特定的蛋白质结构域及其操纵子位点的选择是因为它们已经被彻底地研究和很好地描述了。首先，我们的方法从使用分子建模优化分子组分的几何结构开始。我们这样做是为了在细菌生物体中实现前所未有的可控和反式激活功能的结合。然后，这些设备被建造成激活绿色荧光蛋白的表达。他们独特的功能被发现是强大的紧密和激活表达的基因水平的许多倍，通过流式细胞仪实验测量。用随机动力学模型对装置进行了进一步表征。这里提出的新设备可能成为生物学家用来控制细菌基因表达的分子工具箱的有用补充。所使用的方法也可能是设计、开发和表征这样的装置和更复杂的基因调控网络的文库的基础。
Using an original workflow, we have modeled, constructed, and characterized two new molecular devices that inducibly activate gene expression inEscherichia coli. The devices, prokaryotic-TetOn and prokaryotic-TetOff, were built by fusing an inducible DNA-binding protein domain to a transcription activation domain and constructing a complementary synthetic promoter sequence through which they could control downstream gene expression. In particular, the transactivators were built using variants of the tetracycline repressor, TetR, and the transactivating domain of the LuxR activator. The complementary promoter sequence included TetR’s operator,tetO,and elements of theluxpromoter. These specific protein domains and their operator sites were chosen as they have been thoroughly studied and well characterized. First, our methodology began with optimizing the geometry of the molecular components using molecular modeling. We did so to achieve an unprecedented combination of controllable and transactivating function in bacterial organisms. The devices were then built to activate the expression of green fluorescent protein. Their unique function was found to be robustly tight and activating many-fold increases of expressed gene levels, as measured by flow cytometry experiments. The devices were further characterized with stochastic kinetic models. The new devices presented herein may become useful additions to the molecular toolboxes used by biologists to control bacterial gene expression. The methodology used may also be a foundation for the design, development, and characterization of a library of such devices and more complex gene regulatory networks.
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