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Introduction and RationaleThe detection of bioavailable phenol is a very important issue in environmental and human hazard assessment. Despite modest developments recently, there is a stern need for development of novel biosensors with high sensitivity for priority phenol pollutants. DmpR (Dimethyl phenol regulatory protein), an NtrC-like regulatory protein for the phenol degradation of Pseudomonas sp. strain CF600, represents an attractive biosensor regimen. Thus, we sought to design a novel biosensor by modifying the phenol detection capacity of DmpR by using mutagenic PCR.MethodsBinding sites of ‘A’ domain of DmpR were predicted by LIGSITE, and molecular docking was performed by using GOLD to identify the regions where phenol may interact with DmpR. Total five point mutations, one single at position 42 (Phe-to-Leu), two double at 140 (Asp-to-Glu) and 143 (Gln-to-Leu), and two double at L113M (Leu-to- Met) and D116A (Asp-to- Ala) were created in DmpR by site-directed mutagenesis to construct the reporter plasmids pRLuc42R, pRLuc140p143R, and pRLuc113p116R, respectively. Luciferase assays were performed to measure the activity of luc gene in the presence of phenol and its derivatives, while RT-PCR was used to check the expression of luc gene in the presence of phenol.ResultsOnly pRLuc42R and pRLuc113p116R showed positive responses to phenolic effectors. The lowest detectable concentration of phenol was 0.5 µM (0.047 mg/L), 0.1 µM for 2, 4-dimethylphenol and 2-nitrophenol, 10 µM for 2, 4, 6-trichlorophenol and 2-chlorophenol, 100 µM for 2, 4-dichlorophenol, 0.01 µM for 4-nitrophenol, and 1 µM for o-cresol. These concentrations were measured by modified luciferase assay within 3 hrs compared to 6–7 hrs in previous studies. Importantly, increased expression of luciferase gene of pRLuc42R was observed by RT-PCR.ConclusionsThe present study offers an effective strategy to design a quick and sensitive biosensor for phenol by constructing recombinant bacteria having DmpR gene.

This Letter presents a quantitative in situ scanning electron microscope (SEM) nanoscale high and very high cycle fatigue (HCF/VHCF) investigation of Ni microbeams under bending, using a MEMS microresonator as an integrated testing machine. The novel technique highlights ultraslow fatigue crack growth (average values down to ∼10 m/cycle) that has heretofore not been reported and that indicates a discontinuous process; it also reveals strong environmental effects on fatigue lives that are 3 orders of magnitude longer in a vacuum than in air. This ultraslow fatigue regime does not follow the well documented fatigue mechanisms that rely on the common crack tip stress intensification, mediated by dislocation emission and associated with much larger crack growth rates. Instead, our study reveals fatigue nucleation and propagation mechanisms that mainly result from room temperature void formation based on vacancy condensation processes that are strongly affected by oxygen. This study therefore shows significant size effects governing the bending high/very high cycle fatigue behavior of metals at the micro- and nanoscales, whereby the stress concentration effect at the tip of a growing small fatigue crack is assumed to be greatly reduced by the effect of the bending-induced extreme stress gradients, which prevents any significant cyclic crack tip opening displacement. In this scenario, ultraslow processes relying on vacancy formation at the subsurface or in the vicinity of a crack tip and subsequent condensation into voids become the dominant fatigue mechanisms.

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