Application of fluorescent protein-tagged trans factors and immobilized cis elements to monitoring of toxic metals based on in vitro protein-DNA interactions

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 Pars-Oars-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 mu g/l or Pb/Cd up to 100 mu g/l. Detection limits were 10 mu g/l for As(III) 10 mu g/l for Cd, and 20 mu g/l for Pb with a microplate fluororeader, whereas 5.0 mu g/l for As(III), 1.0 mu g/l for Cd, and 10 mu g/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. (C) 2010 Elsevier B.V. All rights reserved.