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Synthetic Biology Tools for the FastGrowing Marine Bacterium Vibrio natriegens Tanya Tschirhart, Vrinda Shukla, Erin E. Kelly, Zachary Schultzhaus, Erin NewRingeisen, Jeffrey S. Erickson, Zheng Wang, Whitney garcia, Emaleigh Curl, Robert G. Egbert, Enoch Yeung, and Gary J. Vora ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00176 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019
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ACS Synthetic Biology
Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens
Tanya Tschirhart†, Vrinda Shukla‡, Erin E. Kelly§, Zachary Schultzhaus§, Erin NewRingeisen‡, Jeffrey S. Erickson‡, Zheng Wang‡, Whitney Garcia&, Emaleigh Curl#, Robert G. Egbert¶, Enoch Yeungˆ, Gary J. Vora‡*
†American
Society for Engineering Education, Postdoctoral Fellowship Program, US Naval Research Laboratory, Washington, D.C. 20375, United States of America ‡Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, D.C. 20375, United States of America §National Research Council, Postdoctoral Fellowship Program, US Naval Research Laboratory, Washington, D.C. 20375, United States of America ¶Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, United States of America &Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, United States of America #Biomolecular Science and Engineering, University of California Santa Barbara, Santa Barbara CA 93106, United States of America ^Department of Mechanical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, United States of America
*Corresponding Author Gary J. Vora, Ph.D., Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Bldg. 30 / Code 6910, 4555 Overlook Avenue – SW, Washington D.C. 20375, United States of America. Tel: 202.767.0394, Fax: 202.767.9594, E-mail:
[email protected], ORCID iD: 0000-0002-0657-8597
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ABSTRACT: The fast-growing non-model marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly-used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this non-model chassis.
KEYWORDS: Vibrio, marine, UBER, Light Plate Apparatus, optogenetics
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ACS Synthetic Biology
Despite rather impressive advancements in synthetic biology and metabolic engineering, our ability to meaningfully exploit biological systems remains restricted to a relatively small cadre of model microorganisms. In order to fully utilize a broader spectrum of natural and engineered capabilities, a wider variety of microorganisms needs to be explored1-3. One such candidate is Vibrio natriegens, a Gram-negative marine bacterium that was first reported in the literature ~60 years ago (previously classified as Pseudomonas natriegens)4, 5 yet remained sparsely studied. Recently, V. natriegens has seen a surge of interest as two advantageous characteristics, its reported doubling time of
