Development of Fluorescent Bacteria with <i>Lux</i> and Riboflavin Genes
Lumazine protein from marine luminescent bacteria of <i>Photobacterium</i> 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-incr...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-03-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/6/5096 |
| Summary: | Lumazine protein from marine luminescent bacteria of <i>Photobacterium</i> 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 <i>rib</i> operon of <i>Bacillus subtilis</i>, was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-<i>lum</i>P and pRFN4-Pp <i>lux</i>LP N-<i>lum</i>P) were constructed by amplifying the DNA encoding the N-<i>lum</i>P gene (<i>lux</i>L) from <i>P. phosphoreum</i> and the promoter region (<i>lux</i>LP) present upstream of the <i>lux</i> operon of the gene by PCR and ligating into the pRFN4-Pp N-<i>lum</i>P plasmid. A new recombinant plasmid, pRFN4-Pp <i>lux</i>LP-N-<i>lum</i>P, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into <i>Escherichia coli</i>. When this plasmid was transformed into <i>E. coli</i> 43R, the fluorescence intensity of transformants was 500 times greater than that of <i>E. coli</i> alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the <i>lux</i> promoter exhibited expression that was so high as to show fluorescence in single <i>E. coli</i> cells. The fluorescent bacterial systems developed in the present study using <i>lux</i> and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times. |
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| ISSN: | 1661-6596 1422-0067 |