spectrofluorometer fs5 fluorescence spectrometer  (Edinburgh Instruments)

Journal: Frontiers in Chemistry

Article Title: A genetically encoded fluorescent biosensor for sensitive detection of cellular c-di-GMP levels in Escherichia coli

doi: 10.3389/fchem.2024.1528626

Figure Lengend Snippet: The application of cdiGEBS for investigating the effects of chemical compounds on cellular c-di-GMP levels. (A) Schematic illustration for detecting the effects of CSPs on cellular c-di-GMP levels by cdiGEBS. (B) Fluorescence intensities of strains co-expressing different CSPs and cdiGEBS. pCDFDuet vectors expressing each CSP were co-transformed with the cdiGEBS plasmids (pBAD33-MrkH, P mrkA -mScarlet I). The strain harboring the pBAD33-MrkH, P mrkA -mScarlet I, and pCDFDuet-Empty plasmids were used as the “Empty vector” control. (C) Fluorescence emission spectra of strains co-expressing different CSPs and cdiGEBS. Representative curves from three independent measurements are shown. For (B, C) , fluorescence was recorded by using a fluorescence spectrometer with the excitation and emission wavelengths set at 568 nm and 589 nm, respectively. (D, E) Time-dependent fluorescence changes of strains expressing cdiGEBS in the presence of various concentrations of tyrosol (D) and ampicillin (E) . Strains expressing cdiGEBS were cultured in 96-well microplates to allow continuous measurement of fluorescence and optical density using a microplate reader. (F) Fluorescence intensities of strains expressing cdiGEBS after 21 h of treatment with various concentrations of ampicillin. For (B, F) , individual data points (circles) and mean ± SD ( n = 3) are shown. Statistical analysis was performed with GraphPad Prism 8 using one-way ANOVA with Tukey’s multiple comparisons tests.

Article Snippet: Following 6 h of 6 × His-MBP-CSP expression, cells equivalent to 4 OD 600 nm units were harvested and resuspended in 2 mL of PBS buffer for fluorescence intensity and spectral measurements using a fluorescence spectrometer (FS5 Spectrofluorometer, Edinburgh Instruments), with the excitation wavelength set at 568 nm and an emission range of 575–650 nm.

Techniques: Fluorescence, Expressing, Transformation Assay, Plasmid Preparation, Control, Cell Culture