Open AccessEstablishing a Cell-Free Vibrio natriegens Expression System
Author(s) -
Daniel J. Wiegand,
Henry H. Lee,
Nili Ostrov,
George M. Church
Publication year - 2018
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.8b00222
Subject(s) - synthetic biology , metabolic engineering , biology , protein biosynthesis , yield (engineering) , green fluorescent protein , bacteria , ribosomal protein , microbiology and biotechnology , computational biology , cell free protein synthesis , protein expression , biochemistry , gene , ribosome , genetics , rna , materials science , metallurgy
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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