Ptrkh3 slpgfp vector

The pTRKH3-slpGFP vector (Addgene, Watertown, Massachusetts, United States) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert the T7 transcriptional terminator at BamHI/EcoRV sites, and insert the linker sequence containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using In-Fusion method (Clontech, Mountain View, CA, United States). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the L. paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/ml) selection. Both parental plasmid (pLP) and NAPE-PLD expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa, and Sharpe (MRS) broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain) both supplemented with erythromycin 5 μg/ml (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37°C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/ml was obtained through a colony number correction for the dilution factor.

The pTRKH3-slpGFP vector (Addgene, Watertown, MA, USA) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert T7 transcriptional terminators at BamHI/EcoRV sites, and insert linker sequences containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using the In-Fusion method (Clontech, Mountain View, CA, USA). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the Lactobacillus paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/mL) selection. Both parental plasmid (pLP) and NAPE-PLD-expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa and Sharpe (MRS)-broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain), both supplemented with erythromycin 5 μg/mL (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37 °C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/mL were obtained through a colonies number correction for the dilution factor.