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A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis
N Corneau1, C Dubé, G LaPointe
1Département de biochimie, Faculté des sciences et génie, Université Laval, Québec, Canada.
Letters in Applied Microbiology
|July 10, 2001
Summary
This study introduces a coelectroporation method for isolating and characterizing cryptic lactococcal plasmids. The technique efficiently identifies plasmids with unknown functions, aiding in the development of novel food-grade lactococcal strains.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Lactococcal plasmids often have unassigned functions, hindering genetic studies and strain development.
- Efficient methods are needed to isolate and characterize these cryptic plasmids.
Purpose of the Study:
- To evaluate a coelectroporation method for the indirect selection of lactococcal plasmids with unassigned functions.
- To facilitate the isolation and functional assignment of cryptic plasmids.
Main Methods:
- A marker plasmid conferring erythromycin resistance (Eryr) was coelectroporated with cryptic plasmids into a recipient lactococcal strain.
- Optimization of the marker to cryptic plasmid ratio was performed to maximize cotransformation efficiency.
- Erythromycin-resistant transformants were selected, and plasmids were extracted for analysis.
Main Results:
- An average of 20% cotransformants was achieved by optimizing the plasmid ratio.
- Combinations of multiple cryptic plasmids were successfully cotransformed.
- The marker plasmid was readily eliminated from cotransformed cells without selective pressure.
Conclusions:
- The coelectroporation method significantly reduces the screening effort required to identify transformants containing cryptic plasmids.
- This approach aids in assigning functions to unknown plasmids and constructing novel food-grade lactococcal strains with desired plasmid combinations.