A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level
Determination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2015-08-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114317 |
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| author | Angela Glaeser Ralf Heermann |
| author_facet | Angela Glaeser Ralf Heermann |
| author_sort | Angela Glaeser |
| collection | DOAJ |
| description | Determination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically different forms, called the primary and secondary variant. A tool for generating stable genomic integrations of reporter genes has been lacking for these bacteria, and this has hampered the acquisition of reliable data sets for promoter activities at the single-cell level. We therefore generated a plasmid tool named pPINT-mCherry for the easy and stable introduction of gene fragments upstream of an mCherry reporter gene followed by stable integration of the plasmid into the P. luminescens genome at the rpmE/glmS intergenic region. We demonstrate that the genomic integration of reporter genes for single-cell analysis is necessary in P. luminescens since plasmid-borne reporter genes mimic heterogeneity and are therefore not applicable in these bacteria, in contrast to their use in single-cell analysis in other bacteria like Escherichia coli. |
| format | Article |
| id | doaj-art-afa5caa6c7af47caaa493b3eab8c3bcc |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-08-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-afa5caa6c7af47caaa493b3eab8c3bcc2025-08-20T02:26:06ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-08-01592748110.2144/000114317A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell levelAngela Glaeser0Ralf Heermann11Ludwig-Maximilians-Universität München, Biozentrum, Bereich Mikrobiologie, Martinsried/München, Germany1Ludwig-Maximilians-Universität München, Biozentrum, Bereich Mikrobiologie, Martinsried/München, GermanyDetermination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically different forms, called the primary and secondary variant. A tool for generating stable genomic integrations of reporter genes has been lacking for these bacteria, and this has hampered the acquisition of reliable data sets for promoter activities at the single-cell level. We therefore generated a plasmid tool named pPINT-mCherry for the easy and stable introduction of gene fragments upstream of an mCherry reporter gene followed by stable integration of the plasmid into the P. luminescens genome at the rpmE/glmS intergenic region. We demonstrate that the genomic integration of reporter genes for single-cell analysis is necessary in P. luminescens since plasmid-borne reporter genes mimic heterogeneity and are therefore not applicable in these bacteria, in contrast to their use in single-cell analysis in other bacteria like Escherichia coli.https://www.future-science.com/doi/10.2144/000114317phenotypic heterogeneitypromoter activitygenomic integrationPhotorhabdus |
| spellingShingle | Angela Glaeser Ralf Heermann A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level BioTechniques phenotypic heterogeneity promoter activity genomic integration Photorhabdus |
| title | A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level |
| title_full | A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level |
| title_fullStr | A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level |
| title_full_unstemmed | A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level |
| title_short | A novel tool for stable genomic reporter gene integration to analyze heterogeneity in Photorhabdus luminescens at the single-cell level |
| title_sort | novel tool for stable genomic reporter gene integration to analyze heterogeneity in photorhabdus luminescens at the single cell level |
| topic | phenotypic heterogeneity promoter activity genomic integration Photorhabdus |
| url | https://www.future-science.com/doi/10.2144/000114317 |
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