Labeling Of Shewanella Decolorationis S12 With Fluorescent Protein

The aim of this study is to construct genetically engineered strains of Shewanelladecolorationi S12 which are labeled with enhanced yellow fluorescent protein (EYFP)through transposon and site-specific homologous recombination.The plasmid pTE-okm, which was obtained by inserting the eyfp gene into theplasposon pTnMod-okm, transferred from E.coli S17-1 into Shewanella decolorationisS12 through conjugation. The minitransposon within pTE-okm was transposed to thechromosome of Shewanella decolorationis S12 and made the strain survive. Clonesexpressing EYFP was screened out under a fluorescent microscope and it was makesure that pTE-okm suicided within these clones by PCR. One of these clones,named S12-40, had the same growth rate and decoloration ability as that of theoriginal strain S12-1 and it expressed EYFP stably even after re-culturingsuccessively for 20 generations (one generation every 8 hours).After analyzing the genome sequence of Shewanella oneidensis MR-1 from gene bank,an intergene region (about 1.3Kb) between gene S00500 and S00501 was selected asthe site into which the Shewanella decolorationis's potential promoter PazoR andgene eyfp (PazoR-eyfp) was recombianted. The azoR promoter and gene eyfp waslinked together by primer overlapping. Using the same method, the fragment whichwas named PazoR-eyfp was inserted into the intergene region and a recombinantDNA fragment about 1.9Kb was obtained. The fragment was transformed into S12 byelectroporation. Clones expressing EYFP were screened out and one of these cloneswas named S12-SF. The insertion of the 1.9Kb DNA fragment into the specific site onthe genomic DNA through homologous recombination was proved by PCR using S12 -SF total DNA as template. The fluorescent character of S12-SF kept stable aftersuccessively reculturing.This study obtained two strains of genetically engineered strains of Shewanelladecolorationis S12 labeled by EYFP, and laid a foundation for the study on theecological performance of Shewanella decolorationis S12.