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A counterselection method for Lactococcus lactis genome editing based on class IIa bacteriocin sensitivity.

Xing Wan1, Anne M Usvalampi1,2, Per E J Saris1

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This study introduces a novel counterselection method for Lactococcus lactis chromosome engineering. The technique efficiently deletes large DNA fragments using bacteriocin sensitivity and PCR, enabling precise genomic modifications.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Lactococcus lactis is a key industrial bacterium.
  • Efficient methods for chromosomal engineering are crucial for strain improvement.
  • Existing methods for Lactococcus lactis chromosomal deletion can be inefficient.

Purpose of the Study:

  • To develop a new counterselection method for deleting DNA fragments from the Lactococcus lactis chromosome.
  • To create a tool for efficient and precise genomic modifications in Lactococcus lactis.

Main Methods:

  • A non-replicating plasmid vector was designed for integration into the Lactococcus lactis chromosome.
  • The vector contains an erythromycin resistance gene and homologous recombination fragments.
  • Expression of Listeria monocytogenes mptC gene conferred sensitivity to class IIa bacteriocins, used for counterselection.
  • Polymerase Chain Reaction (PCR) was used to confirm desired deletions.

Main Results:

  • Successfully deleted 22-kb and 33-kb chromosomal fragments from Lactococcus lactis strain N8.
  • The method demonstrated high efficiency in generating successive deletions.
  • The counterselection strategy effectively eliminated unwanted integrants.

Conclusions:

  • The presented counterselection method is a convenient, efficient, and inexpensive technique.
  • This method facilitates the generation of successive deletions in the Lactococcus lactis chromosome.
  • The approach is valuable for bacterial genetics and strain development.