Kelwick Richard, Webb Alexander J, MacDonald James T, Freemont Paul S
Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK; Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Metab Eng. 2016 Nov;38:370-381. doi: 10.1016/j.ymben.2016.09.008. Epub 2016 Sep 30.
Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.
无细胞转录-翻译系统最初应用于体外蛋白质生产。最近，合成生物学使这些系统能够在系统设计背景下用于对DNA调控元件、遗传逻辑电路和生物合成途径进行原型设计。革兰氏阳性土壤细菌枯草芽孢杆菌是一种具有重要工业价值的成熟模式生物。为此，我们开发了几种基于枯草芽孢杆菌的无细胞系统。我们改进的基于枯草芽孢杆菌WB800N的系统能够产生0.8μM的绿色荧光蛋白（GFP），与枯草芽孢杆菌168无细胞系统相比，产量提高了约72倍。我们改进的系统用于枯草芽孢杆菌启动子文库的原型设计，在该文库中，我们设计了几个源自野生型P（σA）启动子的启动子，它们在体外和体内表现出一系列相当的转录活性。此外，我们展示了诱导表达系统的无细胞特性以及一种模型酶——海肾荧光素酶的活性。
