A pBBR1-based vector with IncP group plasmid compatibility for Methylorubrum extorquens
- 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.
Author: | Laura PöschelORCiDGND, Elisabeth Gehr, Markus BuchhauptORCiDGND |
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URN: | urn:nbn:de:hebis:30:3-795568 |
DOI: | https://doi.org/10.1002/mbo3.1325 |
Parent Title (English): | MicrobiologyOpen |
Publisher: | John Wiley & Sons Ltd. |
Place of publication: | Malden, Mass. |
Document Type: | Article |
Language: | English |
Year of Completion: | 2022 |
Year of first Publication: | 2022 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2023/11/13 |
Tag: | Methylorubrum extorquens AM1; Rep gene; cotransformation; expression system; plasmid; plasmid copy number |
Volume: | 11 |
Issue: | 5, e1325 |
Article Number: | e1325 |
Page Number: | 9 |
HeBIS-PPN: | 515127434 |
Institutes: | Biowissenschaften |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |