The sequence of red fluorescent proteins (rfps) was obtained by performing rfp-clone-F/R cloning using plasmid pNZ8048-rfp as a template. Afterward, plasmids pLEB124-P45-rfp, pLEB124-P1-rfp, and pLEB124-P2-rfp were constructed by recombination to replace the cat gene in the pLEB124-P45-cat, pLEB124-P1-cat, and pLEB124-P2-cat plasmids, respectively. The newly constructed plasmids were sequenced and individually verified.
The same transformation method was employed to introduce plasmids pLEB124-P45-rfp, pLEB124-P1-rfp, and pLEB124-P2-rfp into the competent cells of L. lactis N8-1 and L. lactis N8-2. Consequently, strains N8-1-P45-rfp, N8-1-P1-rfp, N8-1-P2-rfp, N8-2-P45-rfp, N8-2-P1-rfp, and N8-2-P2-rfp were obtained. The culture was transferred to a 96-well cell culture plate in the logarithmic phase. The plates were then incubated under light-protected conditions. The fluorescence intensity of the strains was measured using ELISA after incubation for 14 h and 20 h. The parameter settings for ELISA were excitation light at 587 nm and scattering light at 610 nm. The fluorescence values obtained from each strain were used to precisely measure the promoter expression.
The same transformation method was employed to introduce plasmids pLEB124-P45-rfp, pLEB124-P1-rfp, and pLEB124-P2-rfp into the competent cells of L. lactis N8-1 and L. lactis N8-2. Consequently, strains N8-1-P45-rfp, N8-1-P1-rfp, N8-1-P2-rfp, N8-2-P45-rfp, N8-2-P1-rfp, and N8-2-P2-rfp were obtained. The culture was transferred to a 96-well cell culture plate in the logarithmic phase. The plates were then incubated under light-protected conditions. The fluorescence intensity of the strains was measured using ELISA after incubation for 14 h and 20 h. The parameter settings for ELISA were excitation light at 587 nm and scattering light at 610 nm. The fluorescence values obtained from each strain were used to precisely measure the promoter expression.
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