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Establishing a cell-free Vibrio natriegens expression system

Daniel J. Wiegand, Henry H. Lee, Nili Ostrov, George M. Church
doi: https://doi.org/10.1101/331645
Daniel J. Wiegand
1Department of Genetics, Harvard Medical School, Boston, MA 02115
2Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115
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Henry H. Lee
1Department of Genetics, Harvard Medical School, Boston, MA 02115
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Nili Ostrov
1Department of Genetics, Harvard Medical School, Boston, MA 02115
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  • For correspondence: nili_ostrov@hms.harvard.edu gchurch@genetics.med.harvard.edu
George M. Church
1Department of Genetics, Harvard Medical School, Boston, MA 02115
2Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115
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  • For correspondence: nili_ostrov@hms.harvard.edu gchurch@genetics.med.harvard.edu
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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 >260µg/mL of super-folder GFP in a small-scale batch reaction after three hours. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and extract protein yield. We observed maximal protein yield at incubation temperatures of 26°C 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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Posted May 26, 2018.
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Establishing a cell-free Vibrio natriegens expression system
Daniel J. Wiegand, Henry H. Lee, Nili Ostrov, George M. Church
bioRxiv 331645; doi: https://doi.org/10.1101/331645
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Establishing a cell-free Vibrio natriegens expression system
Daniel J. Wiegand, Henry H. Lee, Nili Ostrov, George M. Church
bioRxiv 331645; doi: https://doi.org/10.1101/331645

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