Optimization of Western Blot for detection of cellspecific localization of DNA binding protein fromstarved cells (Dps) in Nostoc punctiforme

Sammanfattning:

Cyanobacteria belong to the oldest organisms of our planet. They use photosynthesis to produce ATP and gain biomass from carbon dioxide. The cyanobacteria Nostoc punctiforme is a filamentous bacterium that consists of two different types of cells, vegetative cells and heterocysts. The type of cell it differentiates into depends on the media they grow in. In an ammonium-rich medium, the N.punctiforme consists of vegetative cells that differentiate into heterocysts when in the medium is changed to a low-concentration ammonium medium. The ammonium-binding nitrogenase in the heterocysts does not work in an oxidative environment.

During oxidative stress, N.punctiforme produces Dps (DNA binding protein from starved cells) which protects DNA. In the heterocysts the nitrogenase produces hydrogen as a side product. The hypothesis is that Dps is cell specific. In order to study this protein, a fusion of the promotor of Dps and GFP (Green Flourescent Protein) was constructed. To detect GFP, optimization of a Western Blot (WB) for GFP was performed. Protein samples were analyzed in strains of N.punctiforme. In strain 12A, the production of GFP was visualized but the band was not specific. Several attempts of optimization of the WB procedure were performed, but none of them showed clear specific protein detection in the N.punctiforme strains. Further optimization of the WB protocol is needed.