Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Wiegand, Daniel J.; Lee, Henry H.; Ostrov, Nili; Church, George M.

doi:10.3791/59495

Title: Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Abstract

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 μL batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of > 260 μg/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1-2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Authors:

Wiegand, Daniel J. ^[1]; Lee, Henry H. ^[2]; Ostrov, Nili ^[2]; Church, George M. ^[1]

Harvard Medical School, Boston, MA (United States); Wyss Institute for Biologically Inspired Engineering, Boston, MA (United States)
Harvard Medical School, Boston, MA (United States)

Publication Date:: Thu Mar 14 00:00:00 EDT 2019

Research Org.:: Harvard Medical School, Boston, MA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

OSTI Identifier:: 1528911

Grant/Contract Number:: FG02-02ER63445

Resource Type:: Accepted Manuscript

Journal Name:: Journal of Visualized Experiments

Additional Journal Information:: Journal Volume: 145; Journal ID: ISSN 1940-087X

Publisher:: MyJoVE Corp.

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; Synthetic biology; Vibrio natriegens; cell-free protein expression system

Citation Formats


                    Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, and Church, George M. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens.  United States: N. p., 2019. 
Web.  doi:10.3791/59495.

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                    Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, & Church, George M. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens.  United States.  https://doi.org/10.3791/59495

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                    Wiegand, Daniel J., Lee, Henry H., Ostrov, Nili, and Church, George M. Thu .  
"Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens".  United States.  https://doi.org/10.3791/59495.  https://www.osti.gov/servlets/purl/1528911.

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@article{osti_1528911,

  title        = {Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens},

  author       = {Wiegand, Daniel J. and Lee, Henry H. and Ostrov, Nili and Church, George M.},

  abstractNote = {The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 μL batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of > 260 μg/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1-2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.},

  doi          = {10.3791/59495},

  journal      = {Journal of Visualized Experiments},

  number       = ,

  volume       = 145,

  place        = {United States},

  year         = {Thu Mar 14 00:00:00 EDT 2019},

  month        = {Thu Mar 14 00:00:00 EDT 2019}

}

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Works referencing / citing this record:

Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems
journal, October 2019

Khambhati, Khushal; Bhattacharjee, Gargi; Gohil, Nisarg
Frontiers in Bioengineering and Biotechnology, Vol. 7
DOI: 10.3389/fbioe.2019.00248

Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems
journal, October 2019

Khambhati, Khushal; Bhattacharjee, Gargi; Gohil, Nisarg
Frontiers in Bioengineering and Biotechnology, Vol. 7
DOI: 10.3389/fbioe.2019.00248

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Title: Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

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Citation Formats

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