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bioRxiv · 10.1101/2025.08.27.672616

A reference set of functional plasmids for Vibrio natriegens

Brumwell, S. L.·Esmurria, A.·Crits-Christoph, A.·Kang, S. C.·Gilbert, C.·Lee, H. H.·Ostrov, N.
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Abstract

New microbial hosts with superior phenotypes, such as fast growth, are attractive for research and biotechnology, but often lack systematic evaluation of functional genetic parts. A reference set of working plasmids would increase reproducibility and encourage use of these hosts. Here, we use the POSSUM toolkit, a collection of 23 origins-of-replication and 6 antibiotic markers, to identify functional genetic parts for strains of Vibrio natriegens. We applied this to the wild-type strain ATCC 14048 and an engineered variant NBx CyClone, evaluating 414 combinations of origins of replication and antibiotic selection conditions. We show that both strains support five replicons (pNG2, pSa, pSC101ts, p15A, and RSF1010) with NBx CyClone supporting an extra replicon RK2. The assay can be performed in under a week and is compatible with multiple DNA delivery methods. This work demonstrates the feasibility of rapidly establishing reference information to accelerate the adoption of new microbial hosts.

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Brumwell, S. L., Esmurria, A., Crits-Christoph, A., Kang, S. C., Gilbert, C., Lee, H. H., Ostrov, N.. 2025-08-27. A reference set of functional plasmids for Vibrio natriegens. https://doi.org/10.1101/2025.08.27.672616

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