title: Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions.
abstract: Environmental toxic metals cause serious global public health problems. On-site monitoring protects people from exposure to such harmful elements. In this study, the bacterial transcriptional switches were applied to monitoring of toxic metals. ArsR and CadC, trans factors of Escherichia coli and Staphylococcus aureus, were fused to GFP. The fusion proteins, ArsR-GFP and CadC-GFP, associated with cis elements, P(ars)-O(ars) and P(cad)-O(cad), respectively and dissociated from those upon recognition of As(III) or Pb/Cd. Cell lysates containing ArsR-GFP were pre-incubated with As(III) standard solutions for 15 min and loaded into P(ars)-O(ars)-immobilized microplate wells. Cell lysates containing CadC-GFP were pre-incubated with Pb or Cd solutions and loaded into P(cad)-O(cad)-immobilized wells. The cell lysates were incubated for 15 min and removed from the wells. Fluorescence intensity in the wells dose-dependently decreased in response to As(III) up to 200 μg/l or Pb/Cd up to 100 μg/l. Detection limits were 10 μg/l for As(III) 10 μg/l for Cd, and 20 μg/l for Pb with a microplate fluororeader, whereas 5.0 μg/l for As(III), 1.0 μg/l for Cd, and 10 μg/l for Pb with a handheld fluorometer. This method was available to detect Pb/Cd or As(III) in water containing soil extracts. This is the first demonstration of a simple and rapid fluorometry to detect analytes based on in vitro interaction between a cis element and a trans factor.
authors: Faculty of Agriculture, Utsunomiya University, 350 Minemachi, Utsunomiya 321-8505, Japan.
authors: Yasunari
authors: Y
authors: Kawakami
authors: Mohammad Shohel Rana
authors: MS
authors: Siddiki
authors: Koichi
authors: K
authors: Inoue
authors: Hisae
authors: H
authors: Otabayashi
authors: Kazuyuki
authors: K
authors: Yoshida
authors: Shunsaku
authors: S
authors: Ueda
authors: Hitoshi
authors: H
authors: Miyasaka
authors: Isamu
authors: I
authors: Maeda
mesh_terms: D001151:Arsenic / Q000032:analysis / Q000633:toxicity; D001426:Bacterial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002104:Cadmium / Q000032:analysis / Q000633:toxicity; D004269:DNA, Bacterial / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D004784:Environmental Monitoring / Q000379:methods; D029968:Escherichia coli Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins; D007854:Lead / Q000032:analysis / Q000633:toxicity; D019216:Metals, Heavy / Q000032:analysis* / Q000633:toxicity; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012989:Soil Pollutants / Q000032:analysis* / Q000633:toxicity; D020349:Surface Plasmon Resonance / Q000379:methods; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: C489527:ArsR protein, E coli; D001426:Bacterial Proteins; C080050:CadC protein, Bacteria; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D019216:Metals, Heavy; D011993:Recombinant Fusion Proteins; D012989:Soil Pollutants; D015534:Trans-Activators; D002104:Cadmium; D049452:Green Fluorescent Proteins; D007854:Lead; D001151:Arsenic