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Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA
J K Thompson1, K J McConville, C McReynolds
1Food Science Division (Food Microbiology), Department of Agriculture for Northern Ireland, Belfast, UK. kthompson@alpha1.dani.gov.uk
Letters in Applied Microbiology
|February 5, 1998
Summary
Linearized plasmid DNA transforms Lactobacillus plantarum less efficiently than circular DNA. This finding impacts direct cloning strategies in this species, though successful cloning was achieved using selection.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Plasmid DNA conformation is crucial for efficient transformation in many bacterial species.
- Understanding transformation efficiencies is key for genetic manipulation and cloning.
- Lactobacillus plantarum is a significant bacterium in food and probiotic applications.
Purpose of the Study:
- To investigate the impact of plasmid DNA conformation (linear vs. circular) on electrotransformation efficiency in Lactobacillus plantarum.
- To assess the suitability of linear DNA for direct cloning applications in L. plantarum.
- To evaluate the effectiveness of positive selection for cloning bacterial DNA fragments into L. plantarum.
Main Methods:
- Electrotransformation of Lactobacillus plantarum using linearized and covalently closed circular plasmid DNA (pGKV110).
- Religation of linearized plasmid DNA to create open circular forms.
- Transformation efficiency quantification based on antibiotic resistance.
- Cloning of Lactobacillus helveticus DNA fragments using positive selection.
Main Results:
- Linearized plasmid DNA exhibited a 500-fold lower transformation efficiency compared to covalently closed circular plasmid in L. plantarum.
- Open circular plasmid DNA showed only a marginal improvement in transformation efficiency over linear DNA.
- High background transformation by linear DNA could potentially hinder the identification of successful clones.
- Successful cloning of Lactobacillus helveticus DNA fragments was achieved using the shuttle vector pGKV110 with enhanced chloramphenicol resistance selection.
Conclusions:
- Plasmid conformation significantly affects electrotransformation efficiency in Lactobacillus plantarum, with circular forms being superior.
- The use of linear DNA for transformation in L. plantarum may complicate direct cloning due to high background.
- Positive selection strategies are effective for overcoming challenges and enabling successful gene cloning in L. plantarum, even with less efficient DNA forms.