Journal: PLOS Biology

Article Title: Structure of human glycoprotein 2 reveals mechanisms underlying filament formation and adaption to proteolytic environment in the digestive tract

doi: 10.1371/journal.pbio.3003238

Figure Lengend Snippet: a , Western blotting analysis of GP2 derived from human pancreas (donor 1, before and after elastase cleavage) and small intestines (donors 2–4, without elastase treatment), probed with two GP2 antibodies, anti-GP2 (35–179) and anti-GP2 (111–387). −E, GP2 incubated without elastase; +E, GP2 incubated with elastase; +E’, the same sample as +E, but the loading volume for SDS–PAGE was reduced to one-fifth; Mw, molecular weight. b , Representative negative-stain EM images of (e) GP2 derived from the pancreas. Scale bar represents 50 nm. c , The SDS–PAGE of interaction assays between FITC-FimH L and GP2. The bands represent FITC-FimH L that were visualized with fluorescence. Samples were either washed using centrifugal filters (+ in the row labeled “filter”) or directly analyzed using SDS–PAGE without wash (− in the same row) as controls. d , Representative negative-stain EM images of (e) GP2 incubated with or without FimH L , labeled with immunogold and FimH antibodies. White arrows label GP2 filaments and the black arrow labels immunogold beads. Scale bar represents 50 nm. e , Representative light microscopy images of (e) GP2-mediated E. coli cell aggregation. Scale bar represents 10 μm. All assays were repeated independently at least three times with similar results. The data underlying this figure can be found in .

Article Snippet: E. coli TOP10 competent cells (Yeasen, catalog number 11801ES80) were used in this study for GP2-induced aggregation assay.

Techniques: Western Blot, Derivative Assay, Incubation, SDS Page, Molecular Weight, Staining, Fluorescence, Labeling, Light Microscopy

Journal: bioRxiv

Article Title: Modular Engineering of Thermo-Responsive Allosteric Proteins

doi: 10.1101/2025.05.02.651844

Figure Lengend Snippet: a , Assay schematics. b , c , E. coli transformed with plasmids encoding the indicated AraC-LOV variants and an mRFP reporter were incubated at 37°C or 41°C followed by measurements of reporter fluorescence (RFP) and OD600 in a plate reader ( b ), or flow cytometry ( c ). d , Close-ups of the As LOV2 crystal structure. Mutated residues are marked in red and functionally critical elements are marked in blue. Hydrogen bonds are indicated. PDB-ID: 2V0U. b Data represent the mean +/-SD, n = 3 independent experiments.

Article Snippet: Chemically competent Top10 E. coli cells (Thermo Fisher Scientific) were transformed with the assembled constructs and plated on agar supplemented with the appropriate antibiotic.

Techniques: Transformation Assay, Incubation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: Modular Engineering of Thermo-Responsive Allosteric Proteins

doi: 10.1101/2025.05.02.651844

Figure Lengend Snippet: a, b , E. coli carrying a pBAD-mRFP reporter and AraC wt ( a ) or AraC-LOV C450A ( b ) were incubated for 16 h at 37°C or 41°C in the presence of inducers at the indicated concentration. Expression of the AraC variants is IPTG-inducible, while AraC activity is arabinose-dependent. RFP fluorescence and OD600 were evaluated in a plate reader. c , Photographs of spatial gene expression control. AraC-(LOV) reporter strains were plated on agar and one half of the tray was heated while the other half was kept at room temperature. d , E. coli containing a pBad-mRFP reporter and expressing the indicated AraC variant or a dummy control protein were incubated at 37°C. After 5 hours the temperature was increased to 41°C followed by another 19 hours of incubation. Culture density (OD600) and RFP expression (normalized to OD) were periodically assessed in a plate reader. e , Temperature response profiles of AraC-LOV variants. Samples were prepared as in a but incubated at different temperatures. Data was min-max normalized for each sample (see Supplementary Fig. 6). Data represent the mean +/-SD, n = 3 independent experiments. wt, wild-type.

Article Snippet: Chemically competent Top10 E. coli cells (Thermo Fisher Scientific) were transformed with the assembled constructs and plated on agar supplemented with the appropriate antibiotic.

Techniques: Incubation, Concentration Assay, Expressing, Activity Assay, Fluorescence, Gene Expression, Control, Variant Assay

Journal: bioRxiv

Article Title: Modular Engineering of Thermo-Responsive Allosteric Proteins

doi: 10.1101/2025.05.02.651844

Figure Lengend Snippet: a , Assay schematics. b , c , E. coli cultures expressing either wild-type CAT, no CAT, the indicated CAT-K136-LOV linker variants ( b ) or CAT-K136-LOV variants with point mutations in the LOV domain ( c ) were grown in the presence of chloramphenicol. Samples were incubated at either 37°C or 41°C for 16 hours, followed by measurement of the OD600. d , Serial dilutions of the cultures from c (previously incubated at either 37°C or 41°C) were spotted onto LB-agar supplemented with chloramphenicol and incubated at 37°C overnight before the image was taken. e , Serial dilutions of pre-cultures expressing either wt CAT or CAT-K136-LOV C450A were spotted onto LB-agar supplemented with chloramphenicol and the plates were incubated overnight at the indicated temperature. b , c , Data represent the mean +/-SD, n = 3 independent experiments. wt, wild-type.

Article Snippet: Chemically competent Top10 E. coli cells (Thermo Fisher Scientific) were transformed with the assembled constructs and plated on agar supplemented with the appropriate antibiotic.

Techniques: Expressing, Incubation