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Title: Establishing a Cell-Free Vibrio natriegens Expression System

Abstract

We report 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. Lastly, 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.

Authors:
ORCiD logo [1];  [2];  [2]; ORCiD logo [1]
  1. Harvard Medical School, Boston, MA (United States); Wyss Institute for Biologically Inspired Engineering, Boston, MA (United States)
  2. Harvard Medical School, Boston, MA (United States)
Publication Date:
Research Org.:
Harvard Medical School, Boston, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division
OSTI Identifier:
1529064
Grant/Contract Number:  
FG02-02ER63445
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 7; Journal Issue: 10; Related Information: Supplementary Information: https://pubs.acs.org/doi/suppl/10.1021/acssynbio.8b00222/suppl_file/sb8b00222_si_001.pdf; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Vibrio natriegens; cell free system; protein expression

Citation Formats

Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, and Church, George M. Establishing a Cell-Free Vibrio natriegens Expression System. United States: N. p., 2018. Web. doi:10.1021/acssynbio.8b00222.
Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, & Church, George M. Establishing a Cell-Free Vibrio natriegens Expression System. United States. doi:10.1021/acssynbio.8b00222.
Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, and Church, George M. Thu . "Establishing a Cell-Free Vibrio natriegens Expression System". United States. doi:10.1021/acssynbio.8b00222. https://www.osti.gov/servlets/purl/1529064.
@article{osti_1529064,
title = {Establishing a Cell-Free Vibrio natriegens Expression System},
author = {Wiegand, Daniel J. and Lee, Henry H. and Ostrov, Nili and Church, George M.},
abstractNote = {We report 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. Lastly, 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.},
doi = {10.1021/acssynbio.8b00222},
journal = {ACS Synthetic Biology},
number = 10,
volume = 7,
place = {United States},
year = {2018},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
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Cited by: 3 works
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