qmtq2-atp biosensor  (Addgene inc)

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic representation of the structural domain and three-dimensional (3D) structural models of qmTQ2-ATP biosensor. 3D structure models were generated with Alphafold2 . (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-ATP biosensor in the presence (blue) and absence (black) of 10 mM ATP. (E) The dose-response curve of qmTQ2-ATP biosensor to ATP in solution. The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-ATP biosensor in the presence (filled circle) and absence (open circle) of 10 mM ATP. The data represents means ± SD (n = 5). (G) Specificity of qmTQ2-ATP biosensor to ATP and other nucleotides. Δτ represents the lifetime changes with the presence and absence of nucleotides. The data represents means ± SD (n = 5). (H) Sequential pseudo-color images of HeLa cells expressing qmTQ2-ATP biosensor in response to 20 mM 2-DG. Fluorescence lifetime (τ) with pseudo color, scale bar: 10 μm. (I) Box-whisker plot comparing Δτ in HeLa cells between the untreated control group and the group treated with 2-DG for 30 minutes. Double asterisks indicate p<0.05 by Student’s t-test.

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Generated, Fluorescence, Expressing, Whisker Assay, Control

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic representation of the structural domain and 3D structure models of qmTQ2-cAMP biosensor. 3D structure models by Alphafold2. (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-cAMP biosensor in the presence (blue) and absence (black) of 1 mM cAMP. (E) Dose-responsive curves of qmTQ2-cAMP biosensor for cAMP (blue circles) and cGMP (red squares). The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-cAMP biosensor in the presence (closed circles) and absence (open circles) of 1 mM cAMP. The data represents means ± SD (n = 5). (G-H) Representative images (G) and the time course (H) of fluorescence lifetime changes in response to 50 μM forskolin stimulation in qmTQ2-cAMP biosensor expressing COS7 cells (scale bar: 10 μm) (n = 7). (I-J) Representative images (I) and the time course (J) of fluorescence lifetime changes induced by 100 μM isoproterenol application in qmTQ2-cAMP biosensor expressing COS7 cells (n = 7) (scale bar: 10 μm).

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Fluorescence, Expressing

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Schematic domain structure of the qmTQ2-citrate biosensor. (B–D) The emission and excitation spectra (B), the absorption spectra (C) and the fluorescence decay curve (D) of qmTQ2-citrate biosensor in the presence (blue) and absence (black) of 20 mM citrate. (E) The dose-response curve of the qmTQ2-citrate biosensor to citrate in solution. The data represents means ± SD (n = 5). (F) Effect of pH on the fluorescence lifetime of qmTQ2-citrate biosensor in the presence and absence of 20 mM citrate. The data represents means ± SD (n = 5). (G) Specificity of qmTQ2-citrate biosensor to citrate and other metabolites. Δτ represents the dynamic range obtained with the presence and absence of 20 mM metabolites in TBS buffer. The data represents means ± SD (n = 5). (H) Schematic domain structure of qmTQ2-glucose biosensor. (I–K) The emission and excitation spectra (I), the absorption spectra (J) and the fluorescence decay curve (K) of qmTQ2-glucose biosensor in the presence (blue) and absence (black) of 10 mM glucose. (L) Dose-responsive curve of qmTQ2-glucose biosensor to glucose in solution. The data represents means ± SD (n = 5). (M) Effect of pH on the fluorescence lifetime of qmTQ2-glucose biosensor in the presence and absence of 10 mM glucose. The data represents means ± SD (n = 5). (N) Specificity of qmTQ2-glucose biosensor to monosaccharides and glucose metabolism-related molecules. Δτ represents the dynamic range obtained with the presence and absence of 150 μM these molecules. The data represents means ± SD (n = 5).

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

doi: 10.1101/2024.06.29.601303

Figure Lengend Snippet: (A) Sequential pseudo-color images of HeLa cells co-expressing qmTQ2-cAMP and RCaMP1h biosensors in response to simultaneous 50 µM histamine dihydrochloride and 100 µM isoproterenol treatment. (B–C) Typical time course (B) and average trace (C) of fluorescence lifetime changes of qmTQ2-cAMP and RCaMP1h biosensors in response to simultaneous histamine and isoproterenol stimulation (n = 12). (D–E) Inhibition of histamine induced calcium oscillations by isoproterenol. Time course of HeLa cells stimulated with 10 μM histamine solely (C) or after pre-treatment with 100 μM isoproterenol (D). Thick lines represent the average traces with qmTQ2-cAMP biosensor shown in purple and the RCaMP1h biosensor in gray. and colorful thin lines represent individual traces (n = 15), scale bar:10 μm.

Article Snippet: For the qmTQ2-ATP biosensor cDNA construction, the epsilon subunit of the bacterial F o F 1 -ATP synthase cDNA (Addgene plasmid #113906) was inserted into mTQ2-pRSET-A plasmid at Tyr-145 (between the KpnI and EcoRI restriction sites) through various peptide linkers generated by PCR, using In-Fusion Snap Assembly Master Mix.

Techniques: Expressing, Fluorescence, Inhibition