Chappell James, Freemont Paul
Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.
Methods Mol Biol. 2013;1073:61-74. doi: 10.1007/978-1-62703-625-2_7.
The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) σ(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction.
对核糖体结合位点和转录启动子等DNA调控元件进行表征是合成生物学的一个基本目标。通过监测荧光蛋白的合成来表征此类DNA调控元件是一种常用的确定相对或绝对强度的技术。这些测量结果可与数学模型和计算机模拟相结合，以快速评估DNA调控元件单独和组合时的性能，从而辅助对复杂新型生物装置和系统进行可预测且高效的工程设计。在此，我们通过在大肠杆菌体内以及在大肠杆菌无细胞转录和翻译反应体系中体外监测绿色荧光蛋白（GFP）的合成，来描述大肠杆菌（E. coli）σ(70)转录启动子的构建及相对表征。
