Development of Fluorescent Bacteria with Lux and Riboflavin Genes.

Lumazine protein from marine luminescent bacteria of species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light emission of bacterial luminescent systems is used as a sensitive, rapid, and safe assay for an ever-increasing number of biological systems. Plasmid pRFN4, containing the genes encoding riboflavin from the operon of , was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-P and pRFN4-Pp LP N-P) were constructed by amplifying the DNA encoding the N-P gene (L) from and the promoter region (LP) present upstream of the operon of the gene by PCR and ligating into the pRFN4-Pp N-P plasmid. A new recombinant plasmid, pRFN4-Pp LP-N-P, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into . When this plasmid was transformed into 43R, the fluorescence intensity of transformants was 500 times greater than that of alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the promoter exhibited expression that was so high as to show fluorescence in single cells. The fluorescent bacterial systems developed in the present study using and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times.