Shin Jonghyeon, Noireaux Vincent
School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis, MN 55455, USA.
ACS Synth Biol. 2012 Jan 20;1(1):29-41. doi: 10.1021/sb200016s. Epub 2012 Jan 6.
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 polymerases, 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.
无细胞蛋白质合成正成为一种在体外构建和研究复杂信息过程的强大技术。然而，在试管中构建合成基因回路受到现代无细胞系统转录元件库的严重限制，该系统仅由少数噬菌体调控元件组成。在此，我们报告了用一种可与七种大肠杆菌σ因子配合使用的无细胞表达工具箱构建的合成基因回路及其现象学特征。大肠杆菌内源性全酶E(70)被用作主要转录机制。利用其他六种σ因子、两种噬菌体RNA聚合酶和一组阻遏物构建了基本的回路基序，如多级级联、与门和负反馈回路。回路动力学揭示了全局mRNA周转率和被动竞争诱导的转录调控的重要性。无细胞反应可以使用小型透析反应器或在磷脂囊泡（一种人工细胞系统）中长时间进行。这个工具箱是在体外研究基于转录/翻译的复杂生化系统的独特平台。
