A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens
Abstract Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1‐based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1‐based microbial cell fa...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-10-01
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| Series: | MicrobiologyOpen |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/mbo3.1325 |
| _version_ | 1797990126812397568 |
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| author | Laura Pöschel Elisabeth Gehr Markus Buchhaupt |
| author_facet | Laura Pöschel Elisabeth Gehr Markus Buchhaupt |
| author_sort | Laura Pöschel |
| collection | DOAJ |
| description | Abstract Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1‐based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1‐based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1‐based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B‐based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens. |
| first_indexed | 2024-04-11T08:30:27Z |
| format | Article |
| id | doaj.art-c2a744629c7b41618f7cb157da75c364 |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-04-11T08:30:27Z |
| publishDate | 2022-10-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-c2a744629c7b41618f7cb157da75c3642022-12-22T04:34:32ZengWileyMicrobiologyOpen2045-88272022-10-01115n/an/a10.1002/mbo3.1325A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquensLaura Pöschel0Elisabeth Gehr1Markus Buchhaupt2DECHEMA‐Forschungsinstitut Microbial Biotechnology Frankfurt am Main GermanyDECHEMA‐Forschungsinstitut Microbial Biotechnology Frankfurt am Main GermanyDECHEMA‐Forschungsinstitut Microbial Biotechnology Frankfurt am Main GermanyAbstract Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1‐based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1‐based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1‐based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B‐based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.https://doi.org/10.1002/mbo3.1325cotransformationexpression systemMethylorubrum extorquens AM1plasmidplasmid copy numberRep gene |
| spellingShingle | Laura Pöschel Elisabeth Gehr Markus Buchhaupt A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens MicrobiologyOpen cotransformation expression system Methylorubrum extorquens AM1 plasmid plasmid copy number Rep gene |
| title | A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens |
| title_full | A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens |
| title_fullStr | A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens |
| title_full_unstemmed | A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens |
| title_short | A pBBR1‐based vector with IncP group plasmid compatibility for Methylorubrum extorquens |
| title_sort | pbbr1 based vector with incp group plasmid compatibility for methylorubrum extorquens |
| topic | cotransformation expression system Methylorubrum extorquens AM1 plasmid plasmid copy number Rep gene |
| url | https://doi.org/10.1002/mbo3.1325 |
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