Characterizing and prototyping genetic networks with cell-free transcription-translation reactions
Characterizing and prototyping genetic networks with cell-free transcription-translation reactions
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DOI：
10.1016/j.ymeth.2015.05.020
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发表时间：
2015-09-15
期刊：
影响因子：
4.8
通讯作者：
Lucks, Julius B.
中科院分区：
文献类型：
--
作者：
Takahashi, Melissa K.;Hayes, Clarmyra A.;Lucks, Julius B.
合成生物学的一个中心目标是通过设计用于各种生物技术和医疗应用的合成基因网络来设计细胞行为。设计基因网络的过程通常包括一个迭代的“设计-构建-测试”循环，即构建、表征和改变构成网络的部分和连接，直到达到所需的网络功能。在设计和建造这一循环的部分方面取得了许多进展。然而，网络功能的活体表征的缓慢过程往往限制了测试步骤的时间尺度。无细胞转录-翻译(TX-TL)系统为在细胞中进行这些表征提供了一种简单而快速的替代方法。在这里，我们提供了一个无细胞的TX-TL系统的概述，该系统利用了本地的大肠杆菌TX-TL机制，从而允许对大量的部件和网络进行表征。作为一种演示无细胞TX-TL的有效性的方法，我们展示了两个遗传网络的特征：RNA转录级联和蛋白质调节的非相干前馈环。我们还提供了设计TX-TL实验的指南，以表征新的遗传网络。最后，我们讨论了当前和新兴的无细胞系统的应用。(C)2015 Elsevier Inc.保留所有权利。
A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative 'design-build-test' cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription-t ranslation (TX-TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX-TL system that utilizes the native Escherichia coli TX-TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX-TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX-TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems. (C) 2015 Elsevier Inc. All rights reserved.