Establishing a Cell-Free Vibrio natriegens Expression System,ACS Synthetic Biology

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Establishing a Cell-Free Vibrio natriegens Expression System
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-08-30 00:00:00 , DOI: 10.1021/acssynbio.8b00222
Daniel J. Wiegand _{1,

2} , Henry H. Lee ₁ , Nili Ostrov ₁ , George M. Church _{1,

2}

Affiliation

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.

中文翻译：

建立无细胞弧菌弧菌表达系统

迅速生长的纳特氏弧菌是一种新兴的生物技术微生物宿主。利用其生产性细胞成分可能为快速蛋白质生产和代谢途径或遗传回路的原型开发提供引人注目的平台。在这里，我们报告了V. natriegens的发展无细胞表达系统。我们设计了一种简化的粗提物制备方案，并在3小时后的小规模批量反应中获得了260μg/ mL的超级文件夹GFP。培养条件（包括生长培养基和细胞密度）显着影响提取物的翻译动力学和蛋白质产量。我们在26或30°C的孵育温度下观察到了最大的蛋白质产量，并且通过调节对核糖体稳定性至关重要的离子显示出了更高的产量。这项工作建立了一个初始的纳特氏菌无细胞表达系统，能够对纳氏菌进行生物学研究，并将作为加速代谢工程和合成生物学应用的平台。

更新日期：2018-08-30

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