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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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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. |
format | Online Article Text |
id | pubmed-10209150 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
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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