title: Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter-reporter units in tandem for detection of arsenic.
abstract: Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator-the gene for green fluorescent protein (gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 microg L(-1) As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator-arsR-gfp. Thus, segregation of arsR from the ars promoter/operator-gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.
authors.affiliation: Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, 350 Minemachi, Utsunomiya, Tochigi 321-8505, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Tani
authors.forename: Koichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Yuri
authors.initials: Y
authors.lastname: Tani
authors.forename: Md
authors.initials: M
authors.lastname: Harun-ur-Rashid
authors.forename: Norihiro
authors.initials: N
authors.lastname: Azuma
authors.forename: Shunsaku
authors.initials: S
authors.lastname: Ueda
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yoshida
authors.forename: Isamu
authors.initials: I
authors.lastname: Maeda
mesh_terms: D001151:Arsenic / Q000032:analysis*; D001681:Biological Assay / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D015534:Trans-Activators / Q000235:genetics*
chemical_list: C489527:ArsR protein, E coli; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D001151:Arsenic