Difference between revisions of "Team:Freiburg/Collaborations"
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Revision as of 12:22, 8 September 2015
Collaboration with iGEM team Bielefeld
Bielefeld sends a plasmid based on BBa_I746909 that has a translation enhancing sequence (5’-UTR), and Freiburg sends a plasmid containing turboYFP, a His- and a Halo-Tag. We would like to compare if these parts work in different cell-free proteins synthesis environments.
To test the plasmid BBa_I746909 containing a translation enhancing sequence (5'-UTR) we compared it to our GFPs used for cell-free expression (HA-GFP-His6-His6 and His-GFP-Spy). Both plasmids were treated alike and compared to a sample containing no DNA (negative control) and a dilution series of expressed and purified GFP (positive control). All reactions were performed in triplicates. The samples were expressed for 2 hours at 37°C in a 384-well plate using our own lysate and premix. After expression, a western blot and dot blot were performed.
Collaboration with iGEM team Stockholm
The iGEM Team Stockholm wants to develop a bacterial biomarker for early detection of cancer biomarkers. We thought as we are both working on diagnostic tools it would be great to start a collaboration.
Therefore, we wanted to measure the interaction of one of their affibodies to a Her2-protein with our DiaChip. Team Stockholm expressed their affibody with a His-Tag, so it would bind to our Ni-NTA surface and sent the lysate to us. From R&D-Systems we got the purified Her2-antigen, which is a biomarker for breast cancer. Unfortunately, the antigen also had a His-Tag, so we couldn't measure it on Ni-NTA slides, because it would bind also to the surface and not only to the affibody. This would lead to an increased optical thickness all over the slide and we would not be able to get a signal. If we had spotted the affibody-lysate on an unspecific surface, all other proteins in the lysate would have bound to the surface, and the concentration of affibody on the spot would have been too low. So that wasn't a possibility either.
Our last chance was to spot the antigen on an unspecific surface and flush over the affibody-lysate. Due to the small molecular weight of the affibody (10 kDa) it is really hard to see a binding in the iRIf (detection limit: ~10 kDa) but we still tried.
