Team:NAWI-Graz/Proof
Proteomics
The check if the seven proteins, chromoprotein red/blue, ccdB, mazF, ccdA, mazE and dam methylase were expressed we used SDS-PAGE. It was also important to see, if the proteins especially the toxins were secreted to the media. The chromoproteins can be found at about 25kda, at the size of 32kDa we can find the dam methylase. The ccdB has a weight of about 11kDa.
The two antitoxins and mazE are smaller than 10kDA, when seeing the picture in real we could find small bands lower than 10kDA. Unfortinately no time was left for puring a higher concentrated SDS gel.
Although we cannot be 100% sure that all proteins we needed were expressed by E.coli we can be really sure that our insilico worked. For all composite parts one protein can be found on the gel. In case of the toxin part we found the dam methylase wich was located downstream the toxin. In case of the antitoxin composite part we saw the red chromoprotein on the SDS and also with the coloured colonies.
Figure 10 SDS-PAGE useing the growthmedia from our strains. We used the pure solution and the sperarated media in lower 10kDA and bigger than 10kDA for analysis. We can a little lower than the 35kDA standard the dam methylase. At the size of 25kDA we find the chromoproteins from the differnen strains. At 11kDA the ccdB toxin of the blue strain can be detected.
