Journal: Food Chemistry: Molecular Sciences

Article Title: Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

doi: 10.1016/j.fochms.2026.100357

Figure Lengend Snippet: Tagging a nanobody to the N-terminus of β-gal does not affect its activity. (A) Colonies of dH5α bacteria transformed with a plasmid containing the coding sequence for β-gal with Nb16 fused to its N-terminus via a 4AA (GSHV) linker and plated on an LB/Kan plate with IPTG and X-gal. The plate was photographed 24 h after incubation at 37 °C for 16 h. (B), (C), and (D) are the same as (A), except that the bacteria were induced to express β-gal with Nb16 fused to its N-terminus via a longer flexible peptide (GSGASGSHV), a Strep-tag-containing peptide (GSWSHPQFEKHV), or β-gal with purification tags, respectively.

Article Snippet: Milli-Q water was purified in-house using a Milli-Q Advantage A10 system (Millipore, Bedford, MA, USA) and used throughout. o -Nitrophenyl-β-galactoside (ONPG), Isopropyl β-D-1-thiogalactopyranoside (IPTG), Kanamycin (Kan), and X-Gal were purchased from GoldBio (St Louis, MO, USA).

Techniques: Activity Assay, Bacteria, Transformation Assay, Plasmid Preparation, Sequencing, Incubation, Strep-tag, Purification

Journal: Infection and Immunity

Article Title: Quorum-sensing regulator LsrR modulates avian pathogenic Escherichia coli pathogenicity through direct regulation of cysN

doi: 10.1128/iai.00421-25

Figure Lengend Snippet: ( a ) The LsrR protein was expressed and purified. M: BlueStar Prestained Protein Marker (180 kDa); Line 1: purified LsrR protein flow-through with a size of 60 kDa; Line 2: LsrR positive control; and Line 3: PBS negative control. ( b ) Graphic representation and confirmation of the mutant and complementary strains by PCR. PCR lanes 1–3 confirmed cysN deletion with the cysN internal primer-F/R, and lanes 4–6 confirmed cysN deletion with the cysN external primer-F/R. The primers used for the PCR identification of the cysN gene deletion are also indicated. The amplification of the corresponding target genes indicates success in constructing mutant and complemented strains. ( c ) Bluish colonies on X-gal plates indicated interaction between LsrR and cysN (pUT18C-lsrR-BHT101-pKT25-cysN). In contrast, pUT18C-lsrR-BHT101-pKT25-ompA was used as a negative control (no interaction/whitish colonies) and pUT18C-lsrR-BHT101-pKT25-zip as a positive control (interaction/ bluish colonies). ( d ) LsrR binds to the cysN promoters. The binding ability of LsrR to the cysN promoters was determined by electrophoretic mobility shift assay. An increasing amount of LsrR was incubated with excess Cy5.5-labeled probes. Lane 1: free probe. In each panel, from lanes 2 to 7, the concentration of LsrR was 6, 5, 4, 3, 2, and 2 μM, respectively; the amount of Cy5.5-labeled probes was all 50 fmol (the concentrations were all 5 nM). In lanes 8–9, 200 fmol of unlabeled probes were incubated with the LsrR protein beside the labeled probes. In lane 9, protein FabH was run as a negative control. ( e ) LsrR activates cysN transcription. Relative expression of the cysN gene in the APEC94 strain compared to the APEC94∆lsrR mutant, as determined by qRT-PCR. Gene expression was normalized to the housekeeping gene dnaE , and the relative expression level in the APEC94 was set to 1. Data are presented as the mean ± SEM from three independent biological replicates. Statistical significance was determined by an unpaired t -test (*** P < 0.001). ( f ) DNase I footprinting identified an LsrR-binding site within the cysN promoter. Sequence alignment of this protected region revealed the LsrR conserved binding sequence.

Article Snippet: Following overnight culturing, centrifugation, reconstitution, and PCR detection to confirm the sequence, X-GAL Luria-Bertani (LB) agar (Oxoid, Basingstoke, UK) plates were prepared by adding X-GAL (20 mg/mL).

Techniques: Purification, Marker, Positive Control, Negative Control, Mutagenesis, Amplification, Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Concentration Assay, Expressing, Quantitative RT-PCR, Gene Expression, Footprinting, Sequencing