Journal: bioRxiv

Article Title: Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

doi: 10.1101/768473

Figure Lengend Snippet: (A) Brightfield and epifluorescence microscopy of P. aeruginosa (PAO1 Δ wspF Δ psl P BAD pel ) wild-type and Δ pelF mutant, and B. cereus ATCC 10987 wild-type and Δ pelF mutant biofilms grown for 24 h on poly-L-lysine coated glass coverslips and stained with DAPI, to detect DNA, and Wisteria floribunda lectin (WFL) conjugated to Texas red to detect Pel-like extracellular material. Scale bar = 250 μm. (B) Dot blot of extracellular material from B. cereus ATCC 10987 or the Δ pelF mutant grown for 24 h at 30 °C with shaking, separated into crude cell-associated and secreted fractions. Extracellular material was detected using WFL conjugated to horseradish peroxidase (HRP; left ) or 12-Pel primary antibody with HRP-conjugated secondary antibody ( right ).

Article Snippet: The membrane was blocked with 5% (w/v) bovine serum albumin (BSA) in TBS-T for 1 h at room temperature and probed with 10 μg/mL of WFL-HRP (EY Laboratories) in 2% (w/v) BSA in TBS-T with 0.2 g/L CaCl 2 overnight at room temperature with shaking.

Techniques: Epifluorescence Microscopy, Mutagenesis, Staining, Dot Blot

Journal: bioRxiv

Article Title: Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

doi: 10.1101/768473

Figure Lengend Snippet: (A) Biofilm formation by cdgF deletion and complementation strains assessed by the crystal violet assay. (B) Biofilm formation by cdgE deletion and complementation strains assessed by the crystal violet assay. VC, empty vector control; xyl, xylose. Error bars represent the standard error of the mean of six independent trials. (C) Dot blot of extracellular material from B. cereus ATCC 10987 or the isogenic Δ pelF , Δ cdgE , or Δ cdgF mutants grown for 24 h at 30 °C with shaking, separated into crude cell-associated and secreted fractions. Extracellular material was detected using WFL conjugated to horseradish peroxidase (HRP; left ) or 12-Pel primary antibody with HRP-conjugated secondary antibody ( right ).

Article Snippet: The membrane was blocked with 5% (w/v) bovine serum albumin (BSA) in TBS-T for 1 h at room temperature and probed with 10 μg/mL of WFL-HRP (EY Laboratories) in 2% (w/v) BSA in TBS-T with 0.2 g/L CaCl 2 overnight at room temperature with shaking.

Techniques: Crystal Violet Assay, Plasmid Preparation, Dot Blot