Journal: bioRxiv

Article Title: Cellulase catalysis on cell surfaces using Caulobacter S-layer display

doi: 10.1101/2025.07.28.667093

Figure Lengend Snippet: Graphical abstract illustrating the engineered Caulobacter crescentus S-layer display platform. A plasmid-encoded S-layer protein (RsaA) allows surface display of various cellulases, which synergistically degrade cellulose into glucose to support host growth.

Article Snippet: A Bio-Rad Trans-Blot Turbo system was used to transfer protein from the SDS-PAGE gel to a nitrocellulose membrane and incubated in BioRad Everyday Blocking buffer for 10 min. RsaA proteins were labeled during a 1 hr incubation with Rabbit-C Terminal Anti-RsaA polyclonal antibody (courtesy of the Smit lab) diluted 1:40,000 in TBST, Tris-Buffered Saline with 0.1% Tween-20, followed by another 1 hr incubation with Goat-Anti Rabbit IgG StarBrightTM Blue 700 diluted 1:4,000 in TBST (BioRad 12004161).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Cellulase catalysis on cell surfaces using Caulobacter S-layer display

doi: 10.1101/2025.07.28.667093

Figure Lengend Snippet: (A) Immunoblot with polyclonal anti-RsaA antibodies against whole-cells of JS4038. All lanes are from the same membrane and image. (B) Growth curves (OD600) of JS4038 with and without expressing RsaA or an RsaA:Cex fusion protein. Points represent individual replicates; lines represent a LOESS-smoothed best-fit line. (C) Average % volume density of four replicates of cell clump size in each strain compared to cells expressing RsaA only (% volume density of fusion strain minus % volume density of positive RsaA control at all uM), measured with a MasterSizer instrument.(D) Brightfield phase contrast microscopy images of JS4038 strains (43,000x). (E) Immunoblot with polyclonal anti-RsaA antibodies against surface attached protein fractions of JS4038. All lanes are from the same membrane and image. (F) Transmission electron microscopy images of negatively stained JS4038 strains, imaged at 49Kx mag, 80kV. (G) Alphafold predicted structures of RsaA with the multiple cloning site at amino acid 723 (left), RsaA_Cex_V1 (middle), and RsaA_Cex_V2 (right), visualised with Pymol. (H) Cellulase assay performed on JS4038 expressing RsaA or a RsaA:Cex fusion protein with DNP-Cellobioside. Absorbance at 400 nm (A₄₀₀) was corrected for cell density using A₆₀₀. Shaded area represents the standard error of the mean of 3 biological replicates.

Article Snippet: A Bio-Rad Trans-Blot Turbo system was used to transfer protein from the SDS-PAGE gel to a nitrocellulose membrane and incubated in BioRad Everyday Blocking buffer for 10 min. RsaA proteins were labeled during a 1 hr incubation with Rabbit-C Terminal Anti-RsaA polyclonal antibody (courtesy of the Smit lab) diluted 1:40,000 in TBST, Tris-Buffered Saline with 0.1% Tween-20, followed by another 1 hr incubation with Goat-Anti Rabbit IgG StarBrightTM Blue 700 diluted 1:4,000 in TBST (BioRad 12004161).

Techniques: Western Blot, Membrane, Expressing, Control, Microscopy, Transmission Assay, Electron Microscopy, Staining, Cloning

Journal: bioRxiv

Article Title: Cellulase catalysis on cell surfaces using Caulobacter S-layer display

doi: 10.1101/2025.07.28.667093

Figure Lengend Snippet: Comparison of cellulase activity from surface attached protein vs. that shed into the media. (A, B, C, D) DNP-C cellulase assays with error bars representing standard error of the mean of three biological replicates, of treatment 1) (unwashed JS4038 in PYE), 2) (washed JS4038 in HEPES buffer), 3) (surface attached protein fractions), and 4) (PYE culture media only from treatment 1) respectively. (E, F, G, H) Accompanying immunoblots with polyclonal anti-RsaA antibodies of treatment 1), 2), 3) and 4) respectively. Immunoblot from treatment 2) performed on a separate gel to those from treatment 1), 3) and 4).

Article Snippet: A Bio-Rad Trans-Blot Turbo system was used to transfer protein from the SDS-PAGE gel to a nitrocellulose membrane and incubated in BioRad Everyday Blocking buffer for 10 min. RsaA proteins were labeled during a 1 hr incubation with Rabbit-C Terminal Anti-RsaA polyclonal antibody (courtesy of the Smit lab) diluted 1:40,000 in TBST, Tris-Buffered Saline with 0.1% Tween-20, followed by another 1 hr incubation with Goat-Anti Rabbit IgG StarBrightTM Blue 700 diluted 1:4,000 in TBST (BioRad 12004161).

Techniques: Comparison, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Cellulase catalysis on cell surfaces using Caulobacter S-layer display

doi: 10.1101/2025.07.28.667093

Figure Lengend Snippet: (A) Growth curves (OD600) of WT JS4038 in HMG minimal media with glucose, cellobiose or carboxymethyl-cellulose (CMC) as the sole carbon source. (B-D) Growth curves (OD600) of JS4038 expressing RsaA:Cellulase fusion proteins in HMG with CMC as the sole carbon source. (B) JS4038 expressing RsaA:Cex_V1, RsaA:E1_V2 or a 50:50 mix of both strains. (C) JS4038 expressing RsaA:E1_V2, RsaA:Gluc1C or a 50:50 mix of both strains. (D) JS4038 expressing RsaA:Cex_V1, RsaA:Gluc1C, a 50:50 mix of both strains, or a 33:33:34 mix of both strains with JS4038 expressing RsaA:E1_V2. (E) Heatmap of growth curves in B-D. Rows represent individual strains, with hierarchical clustering visualised on the left. All error bars represent standard error of the mean of three biological replicates.

Article Snippet: A Bio-Rad Trans-Blot Turbo system was used to transfer protein from the SDS-PAGE gel to a nitrocellulose membrane and incubated in BioRad Everyday Blocking buffer for 10 min. RsaA proteins were labeled during a 1 hr incubation with Rabbit-C Terminal Anti-RsaA polyclonal antibody (courtesy of the Smit lab) diluted 1:40,000 in TBST, Tris-Buffered Saline with 0.1% Tween-20, followed by another 1 hr incubation with Goat-Anti Rabbit IgG StarBrightTM Blue 700 diluted 1:4,000 in TBST (BioRad 12004161).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth

doi: 10.3389/fcell.2022.834815

Figure Lengend Snippet: Tgfbr2 signaling in primary DP mediates trigeminal neurite outgrowth in co-culture. (A–C) Thy1-YFP trigeminal neurons were cultured in Transwell filters with 3 μm pores atop primary Tgfbr2 f/f DPCs. Immunofluorescence was performed for the YFP protein using an anti-GFP antibody for highly specific staining of neuronal structures over the entire filter. Maximum projections of 100 μm z-stack confocal microscopy images at 10× were collected and stitched with stitching software. TG neurons demonstrated significantly more outgrowth when co-cultured with DPCs (A) than when cultured alone (C) . Neurite outgrowth was not induced when neurons were co-cultured with DP infected with Ad-Cre-GFP to knock down Tgfbr2 (B) . The scale bar is 1,000 μm for (A–C) . (D) Adenovirus infection was confirmed with GFP expression and normalized to DAPI to confirm equivalent numbers of infected cells between control Ad-GFP and Ad-Cre-GFP DPCs (D,E) The scale bar is 100 μm for (D) . Semi-quantitative PCR confirmed Tgfbr2 KD (F) . Images of axonal outgrowth on the filters were auto-thresholded with FIJI software to remove background fluorescence and the remaining pixels were quantified and normalized to the TGN cultured alone from each experimental set (G) . * p <0.05, ** p < 0.01 by 2-way ANOVA tests with a Tukey’s multiple comparison post-hoc set. (H) Representative images of DPCs from co-culture demonstrating reduced expression of an odontoblast marker, DSPP. (I) Quantification of DSPP normalized to DAPI demonstrates a significant reduction in cells with Tgfbr2 deletion. * p < 0.05 by two-tailed unpaired t -test. All analyses are based on four separate experiments.

Article Snippet: Filters were then incubated at 4°C overnight with a 1:200 dilution of chicken anti-GFP primary antibody (1:200, Aves Lab, GFP-1010) to selectively stain the YFP+ neurons (due to the high conservation between GFP and YFP).

Techniques: Co-Culture Assay, Cell Culture, Immunofluorescence, Staining, Confocal Microscopy, Software, Infection, Knockdown, Expressing, Control, Real-time Polymerase Chain Reaction, Fluorescence, Comparison, Marker, Two Tailed Test