Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli

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Abstract

The DNA encoding the luciferase alpha and beta subunits in the luminous marine bacterium Vibrio harveyi (strain 392) is contained within a 4.0-kilobase HindIII fragment. DNA from V. harveyi was digested with HindIII, and the resulting fragments were inserted into the HindIII site of plasmid pBR322. The recombinant plasmids were introduced by transformation into Escherichia coli RR1. The colonies were supplied with n-decanal, the substrate for the bioluminescence reaction, and 12 colonies (of ca. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, has been studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V. harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene. Using antiluciferase antibody to probe proteins transferred from sodium dodecyl sulfate-polyacrylamide gels to nitro-cellulose paper, we have shown that the E. coli transformants produce luciferase that cross-reacts with antiluciferase antibody and is the same molecular weight as V. harveyi luciferase. No alpha subunit could be detected by using antiluciferase antibody in lysates of a subclone, pTB104, which is identical with pTB7 except for deletion of the beta-subunit gene. Thus, the alpha subunit may be unstable and be degraded unless it is associated with beta. The bioluminescence emission spectra of V. harveyi and of E. coli transformants carrying pTB7 are indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)

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