An E. coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells
An E. coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells
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DOI：
10.1021/sb200016s
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发表时间：
2012-01-01
影响因子：
4.7
通讯作者：
Noireaux, Vincent
中科院分区：
文献类型：
--
作者：
Shin, Jonghyeon;Noireaux, Vincent
无细胞蛋白合成正在成为体外构建和研究复杂信息过程的一种强有力的技术。然而，在试管中工程合成基因电路受到现代无细胞系统转录库的严重限制，这些系统仅由少数噬菌体调节元件组成。在这里，我们报告的构建和现象学表征合成基因电路工程的无细胞表达工具箱，与七个大肠杆菌sigma因子。大肠杆菌内源性全酶E-70被用作主要的转录机制。由其他6个sigma因子、2个噬菌体RNA聚合酶和一组抑制因子构建了多级级联、与门和负反馈回路等基本电路基序。电路动力学揭示了全球mRNA周转率和被动竞争诱导的转录调控的重要性。无细胞反应可以在小型透析反应器或磷脂囊泡（一种人工细胞系统）中进行很长时间。这个工具箱是一个独特的平台，研究复杂的转录/翻译为基础的生化系统在体外。
Cell-free protein synthesis is becoming a powerful technique to construct and to study complex informational processes in vitro. Engineering synthetic gene circuits in a test tube, however, is seriously limited by the transcription repertoire of modern cell-free systems, composed of only a few bacteriophage regulatory elements. Here, we report the construction and the phenomenological characterization of synthetic gene circuits engineered with a cell-free expression toolbox that works with the seven E. coli sigma factors. The E. coli endogenous holoenzyme E-70 is used as the primary transcription machinery. Elementary circuit motifs, such as multiple stage cascades, AND gate and negative feedback loops are constructed with the six other sigma factors, two bacteriophage RNA polyrnerases, and a set of repressors. The circuit dynamics reveal the importance of the global mRNA turnover rate and of passive competition-induced transcriptional regulation. Cell-free reactions can be carried out over long periods of time with a small-scale dialysis reactor or in phospholipid vesicles, an artificial cell system. This toolbox is a unique platform to study complex transcription/translation-based biochemical systems in vitro.