zeiss cy5 filter cube  (Carl Zeiss)

Journal: Gut Microbes

Article Title: Lactulose selectively stimulates members of the gut microbiota, as determined by multi-modal activity-based sorting

doi: 10.1080/19490976.2025.2525482

Figure Lengend Snippet: Identification of translationally-active cells. (a) Schematic representation of the workflow for sorting of translationally active cells using L-azidohomoalanine (AHA) labeling as a marker of cellular translational activity and bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence-activated cell sorting (FACS). , translationally active cells were visualized through Cy5 fluorescence (pink) in microscopy images, while total bacterial cells were stained with DAPI (blue). BONCAT-positive cells were then sorted using fluorescence-activated cell sorting (FACS) based on Cy5 fluorescence intensity (boncat, pink) and DAPI signals (blue). Sorted translationally active fractions represent metabolically active bacteria responding to lactulose, enabling downstream taxonomic identification via 16S rRNA gene sequencing. (b) Principal coordinate analysis (PCoA) ordination of genus-level microbiome profiles. “None (0h)” represents baseline (pre-incubation) microbiomes. “None DAPI (6h)” represents BONCAT-negative FACS-sorted cells after 6 h incubation of no amendment samples. “Lactulose BONCAT (6h)” represents BONCAT-positive FACS-sorted cells after 6 h incubation of lactulose-amended samples. Technical replicates were performed. (c) Change in relative abundance of abundant bacterial genera after 6 h incubation of lactulose-amended samples. Bubble size indicates relative abundance (RA) at 0h. The difference in relative abundance of each genus between BONCAT-positive FACS-sorted cells (lactulose BONCAT 6h) of lactulose-amended samples and BONCAT-negative FACS-sorted cells of no amendment samples after 6 h incubation (none DAPI 6h) was calculated as a normalized and scaled enrichment factor (EF; see materials and methods) and is indicated by bubble color. Genera significantly enriched in individual donors are marked with an asterisk and those significantly enriched across all six donors (as determined with DESeq2) are outlined in black.

Article Snippet: For the labeling reaction, a dye premix was prepared by mixing 1.25 μl of 20 mM CuSO4, 2.50 μl of 50 mM THPTA (tris(3-hydroxypropyltriazolylmethyl) amine), and 0.30 μl of Cy5 alkyne dye (Jena Bioscience, Germany).

Techniques: Labeling, Marker, Activity Assay, Fluorescence, FACS, Microscopy, Staining, Metabolic Labelling, Bacteria, Sequencing, Incubation

Journal: bioRxiv

Article Title: Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch

doi: 10.1101/2025.05.28.656632

Figure Lengend Snippet: ( A ) Single-molecule setup for a NusA colocalization study conducted using NusA-Cy5 and PEC-Cy3. The PEC is immobilized by a biotin-streptavidin roadblock. Binding of NusA-Cy5 to the PEC is monitored through direct excitation of Cy5. ( B , D ) Representative single-molecule trajectories showing NusA-Cy5 binding (pink) to PEC-161 (B) and PEC-225 (D) in the presence of 1 mM glycine. ( C , E ) Binding rate constants k on (C) and k off (E) of NusA-Cy5 in the context of PEC-225 and PEC-161 in the absence and presence of 1 mM glycine. ( F ) SiM-KARTS experimental setup. ( G-J ) Binding of the SiM-KARTS probe targeting the β-β’ interaction in aptamer 2 is monitored through direct excitation of the Cy5 fluorescent dye in the absence and presence of glycine. Representative single-molecule trajectories showing the SiM-KARTS probe binding (pink) to P2.2 of PEC-225 recorded in the absence (G) or presence (I) of 1 mM glycine. Hidden Markov modeling (HMM) is shown above of each trace. ( K , L ) Plots showing the cumulative unbound (K) and bound (L) dwell times of the SiM-KARTS probe in the absence and presence of 1 mM glycine in the context of PEC-225. The association ( k on ) and dissociation ( k off ) rate constants of the SiM-KARTS probe are indicated. The reported errors are the error of the fit. The total number of molecules analyzed for each condition is: (−) Glycine = 312; (+) Glycine = 348. (M) Overall binding rate constants ( k on ) of the SiM-KARTS probe in the context of PEC-161, PEC-225, RT-196 and RT-229 constructs determined in the absence (purple) and presence (green) of 1 mM glycine. Error bars are the SD of the mean of independent replicates. The statistical significance of differences was determined using the two-tailed Student’s t-test. a.u., arbitrary unit.

Article Snippet: The azide-carrying ECs were incubated with 500 µM DBCO-Cy5 (Jena Bioscience) in the transcription buffer for 1 h at 37°C.

Techniques: Binding Assay, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch

doi: 10.1101/2025.05.28.656632

Figure Lengend Snippet: (A) smFRET experimental setup. The locations of donor (Cy3 – green) and acceptor (Cy5 – red) fluorophores are indicated. ( B-M ) The following plots are shown for PEC-225 in the presence of 1 mM Mg 2+ (B, C, D), 1 mM Mg 2+ and 100 mM KCl (E, F, G), 1 mM Mg 2+ and 1 mM glycine (H, I, J), and 1 mM Mg 2+ , 100 mM KCl and 1 mM glycine (K, L, M): representative dynamic donor-acceptor (top) and smFRET (bottom) traces (B, E, H, K), smFRET histograms (C, F, I, L), and transition occupancy density plots (TODPs) to capture both dynamic and static molecular behaviors (D, G, J, M). The riboswitch conformational state corresponding to each FRET value is indicated as undocked (U) or docked (D). The SD values of each FRET value and total number of traces (N) are shown. TODPs represent dynamic traces as “off-diagonal” and the static traces as “on-diagonal” contour, where the color scale shows the prevalence of each population.

Article Snippet: The azide-carrying ECs were incubated with 500 µM DBCO-Cy5 (Jena Bioscience) in the transcription buffer for 1 h at 37°C.

Techniques: