A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens.
A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.
Tang
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Department of Biological Sciences, National University of Singapore, 10 Science Drive 4, Singapore, Singapore.
Lu
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Pan
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D016960:Agrobacterium tumefaciens / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase