合成生物学
代谢工程
蛋白质生物合成
无细胞蛋白质合成
绿色荧光蛋白
产量(工程)
细菌
细胞生物学
核糖体蛋白
生物
计算生物学
生物化学
基因
核糖体
遗传学
核糖核酸
冶金
材料科学
作者
Daniel J. Wiegand,Henry H. Lee,Nili Ostrov,George M. Church
标识
DOI:10.1021/acssynbio.8b00222
摘要
The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved >260 μg/mL of superfolder GFP in a small-scale batch reaction after 3 h. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and protein yield of extracts. We observed maximal protein yield at incubation temperatures of 26 or 30 °C, and show improved yield by tuning ions crucial for ribosomal stability. This work establishes an initial V. natriegens cell-free expression system, enables probing of V. natriegens biology, and will serve as a platform to accelerate metabolic engineering and synthetic biology applications.
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