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The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools an...

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Autores principales: Elston, Katherine M., Phillips, Laila E., Leonard, Sean P., Young, Eleanor, Holley, Jo-anne C., Ahsanullah, Tasneem, McReynolds, Braydin, Moran, Nancy A., Barrick, Jeffrey E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10209150/
https://www.ncbi.nlm.nih.gov/pubmed/37225918
http://dx.doi.org/10.1038/s43705-023-00255-3
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author Elston, Katherine M.
Phillips, Laila E.
Leonard, Sean P.
Young, Eleanor
Holley, Jo-anne C.
Ahsanullah, Tasneem
McReynolds, Braydin
Moran, Nancy A.
Barrick, Jeffrey E.
author_facet Elston, Katherine M.
Phillips, Laila E.
Leonard, Sean P.
Young, Eleanor
Holley, Jo-anne C.
Ahsanullah, Tasneem
McReynolds, Braydin
Moran, Nancy A.
Barrick, Jeffrey E.
author_sort Elston, Katherine M.
collection PubMed
description Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli, a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying microbial ecology and host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.
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spelling pubmed-102091502023-05-26 The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae Elston, Katherine M. Phillips, Laila E. Leonard, Sean P. Young, Eleanor Holley, Jo-anne C. Ahsanullah, Tasneem McReynolds, Braydin Moran, Nancy A. Barrick, Jeffrey E. ISME Commun Article Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli, a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying microbial ecology and host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species. Nature Publishing Group UK 2023-05-24 /pmc/articles/PMC10209150/ /pubmed/37225918 http://dx.doi.org/10.1038/s43705-023-00255-3 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Elston, Katherine M.
Phillips, Laila E.
Leonard, Sean P.
Young, Eleanor
Holley, Jo-anne C.
Ahsanullah, Tasneem
McReynolds, Braydin
Moran, Nancy A.
Barrick, Jeffrey E.
The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title_full The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title_fullStr The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title_full_unstemmed The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title_short The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae
title_sort pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of drosophila-colonizing orbaceae
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10209150/
https://www.ncbi.nlm.nih.gov/pubmed/37225918
http://dx.doi.org/10.1038/s43705-023-00255-3
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