Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers.
A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.
Sévigny
P
P
Biotechnology Research Institute, National Research Council Canada, Montréal.
Gossard
F
F
D003001:Cloning, Molecular; D004261:DNA Replication; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008163:Luminescent Measurements; D015183:Restriction Mapping; D014733:Vibrio / Q000235:genetics*