Part:BBa_K2139002:Experience

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Applications of BBa_K2139002

To confirm expression of surface layer fusion protein, C. crescentus cultures were grown and SDS-PAGE and western blot analysis were performed on surface proteins from low pH extraction and on the cell lysates. SDS-PAGE was done using 8% separating gels. Coomassie Brilliant Blue staining and western immunoblotting was performed. Western blots were probed with primary rabbit anti-RsaA polyclonal antibodies at 1/30,000 dilution. Goat anti-rabbit IgG was used as secondary antibody at 1/50,000 dilution. Fluorophore was detected by Odyssey Infrared Imaging System.

Figure 1. (top) Western Blot of C. crescentus cellulase expressing strains, ran on SDS-PAGE and blotted with anti-RsaA. Left to right: (1) Thermofisher ladder, (2) Gluc1C cell lysate, (3) Endo5A cell lysate, (4) E1_399 cell lysate, (5) E1_422 cell lysate, (6) negative control ΔGCSS (ΔrsaA) cell lysate, (7) P4A723 (wildtype) cell lysate, (8) Thermofisher ladder, (9) Gluc1C low pH extracted proteins, (10) Endo5A low pH extracted proteins, (11) E1_399 low pH extracted proteins, (12) E1_422 low pH extracted proteins, (13) ΔGCSS (ΔrsaA) low pH extracted proteins, (14) P4A723 (wildtype) low pH extracted proteins. (bottom) SDS-PAGE stained with coomassie brilliant blue, samples loaded in the same order as above for the Western Blot.

150 uL of each two day old culture culture was aliquoted into a clear 96 well plate(Corning) and 150 μL of assay mix (0.1 mg/ml DNPC in 50 mM pH 5.5 potassium acetate buffer) was added to the each well. OD 400 nm and OD 600 nm was measured every 30 minutes for several hours by a VarioScan plate reader(Thermo). Between measurements the culture was incubating at 30°C. Data was normalized by dividing the OD400nm measurement by the OD600nm measurement.

Figure 2. Assay for cellulase activity with DNPC substrate.

Triplicate 5mL PYE-CM starter cultures of P4A723 (ΔrsaA C. crescentus complemented with wildtype ΔrsaA in p4A723), E1_399, E1_422, Gluc1C and Endo5A were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate containing M2 with 0.2% w/v carboxymethylcellulose (CMC).

Figure 3. Cellulase activity and growth assay results for C. crescentus displaying cellulases compared to C. crescentus expressing wildtype RsaA (P4A723).

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