Establishing a Cell-Free Vibrio natriegens Expression System

ACS Synthetic Biology
Daniel J WiegandGeorge M Church

Abstract

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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Citations

Jun 30, 2019·Applied and Environmental Microbiology·Eugen PfeiferJulia Frunzke
Jun 6, 2019·Methods and Protocols·Nicole E GregorioJavin P Oza
Nov 30, 2019·Nature Reviews. Genetics·Adam D SilvermanMichael C Jewett
Jul 7, 2020·Frontiers in Bioengineering and Biotechnology·Lingkai ZhangJian Li
Apr 22, 2020·Nature Communications·Olivier BorkowskiJean-Loup Faulon
Sep 29, 2020·Frontiers in Microbiology·Man YinZhihu Zhao
Dec 22, 2020·Synthetic Biology·Ashty S KarimMichael C Jewett
Mar 9, 2021·Frontiers in Microbiology·Jiaqi XuLirong Yang
Mar 16, 2021·Frontiers in Bioengineering and Biotechnology·Eden Ozer, Lital Alfonta
Aug 11, 2020·Synthetic and Systems Biotechnology·Stephanie D ColeMatthew W Lux
Apr 10, 2021·Essays in Biochemistry·Felix Thoma, Bastian Blombach
May 20, 2021·Biotechnology Letters·Isabel ThieleSebastian L Riedel
May 22, 2021·ACS Synthetic Biology·Yun-Nam ChoiJeong Wook Lee
Jun 23, 2020·Journal of Controlled Release : Official Journal of the Controlled Release Society·Peter J VoorheesSamuel K Lai
Aug 17, 2019·ACS Synthetic Biology·Tanya TschirhartGary J Vora
Aug 7, 2019·ACS Synthetic Biology·Stephanie D ColeMatthew W Lux
Sep 15, 2020·ACS Synthetic Biology·Sung Sun YimHarris H Wang

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