Journal: Gut Microbes

Article Title: Recombinant production of a diffusible signal factor inhibits Salmonella invasion and animal carriage

doi: 10.1080/19490976.2023.2208498

Figure Lengend Snippet: Stable production of c2-HDA by E. coli reduces expression of Salmonella invasion genes and cell invasion. A. rpfF of C. turicensis under the control of promoters of different strengths (P3 promoter for high expression; P2 promoter for medium expression) was integrated into the genome of E. coli Nissle 1917, and strains were grown in co-culture with S. Typhimurium carrying a plasmid-borne hilA-luxCDABE reporter fusion. hilA expression was measured by luminescence as a function of culture turbidity. Error bars show standard deviation ( n = 5). B. The ratio of Salmonella to E. coli after 24 hours of co-culture was determined by selective plating on MacConkey lactose agar ( n = 3). C. LC-MS was used to analyze fatty-acid extracts of culture supernatants from E. coli strains expressing rpfF , with both high- and medium-expressing constructs demonstrating a peak at 17 minutes, consistent with c2-HDA. The c2-HDA panel is identical to that shown in Fig. 1B, reproduced here for reference. D. Salmonella invasion was determined by measuring its penetration of cultured HEp-2 epithelial cells when grown in co-culture with E. coli expressing rpfF under the control of the P3 promoter or an isogenic strain lacking rpfF . The ratio of Salmonella to E. coli in the inoculum is shown for each strain combination. Salmonella grown alone with the addition of either 20 µM c2-HDA or an equal volume of DMSO were used as controls. Results are shown as the proportion of invasion as compared to Salmonella alone grown with DMSO; mean invasion of this strain was 2.4% of the inoculum. Error bars show standard deviation ( n = 4).

Article Snippet: Mutant strains were constructed using the one-step inactivation method as previously described. rpfF integrated into the E. coli chromosome was cloned under the control of the P3 promoter or the P2 promoter, reported to provide high and medium expressions, respectively, obtained from Addgene.

Techniques: Expressing, Control, Co-Culture Assay, Plasmid Preparation, Standard Deviation, Liquid Chromatography with Mass Spectroscopy, Construct, Cell Culture

Journal: bioRxiv

Article Title: Selective endocytosis controls slit diaphragm maintenance and dynamics

doi: 10.1101/2022.03.01.482547

Figure Lengend Snippet: (A) Shown is a schematic that indicates the genome editing strategy of introducing a myc-tag into the extracellular domain of sns, the fly nephrin. While myc is targeted to the border of exon 2, a marker (in reverse orientation) is inserted into the flanking intron. The marker expresses Dsred under control of the P3 promoter for identification of genome edited flies, but is removable by flanking loxP sites. (B-C’’) Shown are a tangential section (B-B’’) and a cross section (C-C’’) of a garland cell nephrocyte that carries Myc-tag in frame within the locus of fly nephrin, stained for Myc and Neph1 (Kirre). The Myc staining reveals a highly specific staining in a typical fingerprint-like pattern and colocalizes with endogenous fly Neph1. Nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (D-D’’) Silencing fly nephrin abrogates the specific signal from Myc-staining, confirming that the Myc staining indeed reflects endogenously expressed Myc-nephrin. (E) Schematic drawing of the areas used for the quantitation in and (membrane= orange and subcortical area= blue).

Article Snippet: DsRed cDNA under P3 promoter flanked by loxP sites was derived from pHD-DsRed (Addgene plasmid #51434) and placed into the flanking intron that preceded the downstream homology.

Techniques: Marker, Control, Staining, Quantitation Assay, Membrane