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A pBBR1-based vector with IncP group plasmid compatibility for Methylorubrum extorquens
Laura Pöschel1,2, Elisabeth Gehr1, Markus Buchhaupt1
1DECHEMA-Forschungsinstitut, Microbial Biotechnology, Frankfurt am Main, Germany.
Microbiologyopen
|October 31, 2022
Summary
A new plasmid, pMis1_1B, enhances genetic tools for Methylorubrum extorquens, a key microbe in C1-based microbial cell factories. This improved plasmid offers higher transformation rates and expands synthetic biology applications.
Area of Science:
- Microbiology
- Synthetic Biology
- Genetic Engineering
Background:
- Plasmids are crucial genetic tools for research and biotechnology, enabling rapid genetic manipulation.
- Methylorubrum extorquens is a model methylotroph utilized in developing C1-based microbial cell factories.
- Existing plasmid systems, like pCM, have limitations in compatibility and efficiency.
Purpose of the Study:
- To develop a novel pBBR1-based plasmid compatible with the pCM system for Methylorubrum extorquens.
- To improve transformation efficiency and reduce growth defects associated with pBBR1-based plasmids.
- To investigate the potential for enhanced reporter gene expression through co-expression with existing plasmids.
Main Methods:
- Construction and characterization of a novel pBBR1-based plasmid (pMiS1) for M. extorquens.
- Isolation and analysis of suppressor mutants to identify improved plasmid variants (pMis1_1B).
- Assessment of transformation rates, growth phenotypes, and plasmid copy numbers.
- Evaluation of reporter gene expression (mCherry) in co-transformed strains.
Main Results:
- The novel plasmid pMis1_1B demonstrated significantly higher transformation rates and reduced growth defects compared to the initial pMiS1.
- A mutation in the rep gene promoter was identified as responsible for the improved characteristics of pMis1_1B.
- Co-transformation of pMis1_1B with pCM160 was successful, although it induced stronger growth defects.
- Strains co-harboring pCM160 and a pMis1_1B derivative with a mCherry reporter showed enhanced fluorescence, suggesting increased plasmid copy number.
Conclusions:
- The developed pMis1_1B plasmid represents a valuable addition to the genetic toolbox for Methylorubrum extorquens.
- This plasmid facilitates improved genetic manipulation and expands the capabilities for constructing C1-based microbial cell factories.
- Co-expression strategies may lead to enhanced reporter gene expression, warranting further investigation into plasmid interactions and copy number regulation.
