dialysis buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: ( A ) Growth curves of V. cholerae El Tor C6706 (WT) with and without ectopic expression of gp15 in the presence and absence of 50 µg/mL SMX. ( B ) Growth curves of V. cholerae Δ dncV Δ capV (ΔCBASS) mutants with and without ectopic expression of gp15 in the presence and absence of 50 µg/mL SMX. ( C ) Growth curves of V. cholerae WT with and without ectopic expression of gp14 , gp15 , or gp16 in the presence and absence of 50 µg/mL SMX.

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Expressing

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: Conservation and specificity of Gp15 antagonism of CBASS. (A) Homologs of Gp15 ICP3 (YP_004251288.1) were identified by searching against the NCBI protein database using BLASTP. Over 400 hits were identified, and their sequences were retrieved (cutoff E value <1E-20 and coverage >70%). The phylogenetic tree of these Gp15 homologs is built with COBLAT 26 . (B) Structural homology between Gp15 T7 (left, cyan, PDB 7k5c_A,), Gp15 Mak (middle, orange, predicted by Alphafold 3), and Gp15 ICP3 (right, magenta, predicted by Alphafold 3). Structural alignment was performed by PyMOL. ( C ) Growth of E. coli strains in the presence of 50 µg/mL SMX carrying a plasmid with and without the CBASS operon from E. coli TW11681, and in the absence and presence of Gp15 from T7 or coliphage Mak. ( D ) T2 infection efficiency on different E. coli strains with and without CBASS and GP15 ICP3 or Gp15 MAK . The experiment was repeated more than three times, and representative graphs are shown.

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Plasmid Preparation, Infection

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: GP15 inhibits CapV hydrolase activity. (A) Intracellular concentration of cGAMP in V. cholerae Δ dncV Δ capV mutants with and without ectopically expressing dncV and with and without co-expressing gp15 of ICP3. cGAMP levels in the samples were determined by LC-MS/MS using a cGAMP standard curve and normalized to the viable cell counts. A pairwise comparison was performed with a t -test. (* p =0.0286). ( B ) Top: CapV activation level determined by FP-Rh labeling in the presence of increasing concentrations of Gp15. Bottom: The fluorescent signals were quantified and plotted against the ratio between CapV and Gp15. In each reaction, both CapV and cGAMP were used at 0.7 µM, and Gp15 was used from 0 to 1.1 µM. ( C ) Top: CapV activation level determined by FP-Rh labeling in the presence of cGAMP after different treatments. The input cGAMP was pretreated with Gp15 or CapV, and the proteins were removed by Ni-NTA beads. Bottom: The fluorescent signals were quantified. In all cases, each experiment was repeated three times, and representative results are shown with multiple technical replicates (n ≥ 3).

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Activity Assay, Concentration Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Comparison, Activation Assay, Labeling

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: GP15 inhibits CapV not by targeting cGAMP. (A) CapV activation level determined by FP-Rh labeling in the presence of cGAMP after different treatments. The input cGAMP was either untreated (no protein) or pretreated with Gp15 alone, BSA alone, CapV and BSA together, or CapV and Gp15 together, and the proteins were removed by filtering through a 10 KDa MWCO filter. The fluorescent signals of the gel were then quantified. ( B ) The cGAMP levels in the samples shown in (A) were quantified by LC-MS/MS. ( C ) CapV activation level determined by FP-Rh labeling. CapV was activated by cGAMP without Gp15 (left), or CapV was activated by cGAMP after addition of Gp15 (middle), or CapV was activated by cGAMP before the addition of Gp15 (right). Each experiment was repeated three times, and representative results are shown with multiple technical replicates (n ≥ 3).

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Activation Assay, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: Gp15 and CapV directly interact within bacterial cells. ( A ). The BACTH system was used to study the interaction between Gp15 and CapV. E. coli strains co-expressing different Gp15 and CapV T18/T25 fusion proteins were plated on a medium containing X-Gal. EV denotes an empty vector. ( B ) Quantitation of the level of β-galactosidase in different strains co-expressing different CapV or Gp15 fusions. Results were analyzed by ANOVA (**** p < 0.0001 ) ; the assays were repeated twice, and representative results with multiple technical replicates are shown (mean ± SD).

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Expressing, Plasmid Preparation, Quantitation Assay

Journal: bioRxiv

Article Title: A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

doi: 10.1101/2025.07.23.666291

Figure Lengend Snippet: Gp15 cellular level is in excess of CapV during phage infection. (A) Western blot using anti-Gp15 antibodies with cell lysates prepared at different time points after ICP3 infection of V. cholerae WT. The Gp15 standard curve was prepared by mixing different amounts of purified His 6 -Gp15 with V. cholerae WT cell lysate without infection. ( B ) Quantitation of Gp15 amount per cell during ICP3 phage infection. ( C ) Western blot using anti-CapV antibodies with cell lysates from V. cholerae wild type and a strain ectopically overexpressing CapV. The CapV standard curve was prepared by mixing different amounts of purified His 6 -CapV with cell lysates prepared from a strain lacking CapV.

Article Snippet: The CapV activation assay contains 1 μl of diluted CapV Vc -His 6 , 0 to 7 μl His 6 -Gp15 ICP3 (2.1 μM, in 1 μl increments), 7 to 0 μl Gp15 dialysis buffer, 1 μl 3’3’-cGAMP (10 μM in water, InvivoGen), and 4 μl FP-Rh buffer.

Techniques: Infection, Western Blot, Purification, Quantitation Assay