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

Mercury (II) sensor based on monitoring dissociation rate of the trans-acting factor MerR from cis-element by surface plasmon resonance

“…The cell extracts were used to construct a DNA-protein complex on solid surface. ArsR-GFP and CadC-GFP were encoded in pETarsRgfp and pETcadC-gfp, respectively (Kawakami et al, 2010).…”

“…Preparation of cis-elements and cell extracts containing trans-acting factor P ars -O ars -50 (Kawakami et al, 2010) for ArsR-GFP, O ars -30-down (Siddiki et al, 2011) for ArsR-GFP, and P cad -O cad (Kawakami et al, 2010) for CadC-GFP were used as cis-elements. A biotinylated oligonucleotide Pmer-F-3B and its complementary oligonucleotide Pmer-R (Table S1) were used to prepare the cis-element P mer for MerR-GFP or MerR.…”

“…A biotinylated oligonucleotide Pmer-F-3B and its complementary oligonucleotide Pmer-R (Table S1) were used to prepare the cis-element P mer for MerR-GFP or MerR. Double stranded DNA including cis-element was immobilized on solid surface by making biotinylated double stranded DNA contact with streptavidin-coated surface (Kawakami et al, 2010).…”

“…E. coli extracts were prepared by culturing the cells in LB medium at 25°C overnight, followed by disrupting those with an ultrasonic cell disruptor as described previously (Kawakami et al, 2010). The cell extracts were used to construct a DNA-protein complex on solid surface.…”

“…It has been shown that the green fluorescent protein (GFP)-tagged trans-acting factors, ArsR-GFP and CadC-GFP, are applicable to detection of As (III)/Sb (II) and Cd (II)/Pb (II)/Zn (II) by measuring fluorescence intensity of the proteins bound to (Kawakami et al, 2010) and dissociated from the immobilized cis-elements (Siddiki et al, 2011(Siddiki et al, , 2012a(Siddiki et al, , 2012b. When application of the same strategy as established in ArsR and CadC to Hg (II) detection by MerR is considered, no significant quantitative changes of bound and dissociated proteins could be expected because in the mechanism of transcriptional regulation, both the apo and Hg-bound forms could associate to DNA.…”