Molecular cloning and expression of the luciferase coding genes of Vibrio fischeri
Vibrio fischeri is a symbiotic marine bacterium and a nonpathogenic member of the Vibrionaceae which produces luminescence by expressing the lux operon. The lux operon encoding luciferase (luxAB) and proteins required to synthesize the aldehyde substrate (luxCDE), is controlled with luxR and luxI. In this study, we amplified the chromosomal fragment contains luxAB of V. fischeri, amplified fragment cloned into the pTZ57R vector and sequencing to confirmed the fragment. The sub cloning of luxAB gene was carried out in the pETDuet-1 expression vector and expression procedures were performed in Escherichia coli strain Nova blue. As a result, a 2046 bp fragment which contains the whole fragment of luciferase coding genes and intergenic sequences were cloned in pETDuet-1 expression vector. pETDuet luxAB recombinant plasmid was confirmed by restriction analysis; subsequently 76 kD expressed protein was detected using SDS–PAGE and western blot using specific polyclonal antibody. In this study, cloning of the luciferase coding genes was performed successfully, in which the synthesized construct can be applied as a reporter cassette in prokaryotic systems and as a marker or tag in the manipulation, and the control of gene expression in the fields of research, production, control of microorganism and other biotechnological applications.
Key words: Vibrio fischeri, lux operon, luciferase, luminescence.