Protocol for the generation and purification of minicells from Lactiplantibacillus plantarum

초록

Minicells, which are anucleate cells generated by irregular cell division, are emerging aspromising drug delivery systems owing to advances in synthetic biology. However, their developmentis largely limited to a few model bacteria, highlighting the need to explore minicellplatforms in alternative hosts. Lactiplantibacillus plantarum (L. plantarum), a probioticbacterium classified as Generally Recognized as Safe, is an ideal candidate for such exploration. Minicell-producing L. plantarum was engineered by deleting the putative minD genevia plasmid-mediated homologous recombination, which inactivates cell division to formspherical minicells. Anucleate cells were isolated through differential centrifugation and filtration,followed by additional drug treatment to completely eliminate progenitor cells. Microscopyand flow cytometry analyses of the purified sample confirmed the absence of progenitorcells by DAPI staining. This protocol effectively produces bacterial minicells from L. plantarum for use in various biotechnological applications, including therapeutic agent delivery.

키워드