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Team:Freiburg Cell Culture
From 2008.igem.org
Introduction:
Besides the project with the artificial receptor system, it was also tried to activate t-cells (B12.7.5) and b-cells (j558lδmmb1nfleck) with the NIP-linked DNA-origami. Both cell-types have a anti NIP Fab-fragment linked to their receptor. During these tries many problems were faced which could emerge as obstacles in the main project, the artificial receptor which is expressed by 293t-cells.
TThe first problem was the media. The cells were normally kept in Ringer solution during the first measurements which contains too little Mg2+ to keep the origamis stable. So the Mg2+ tolerance of the cells had to be tested in the hope the origamis would be stable if we add Mg2+ to the buffer.
Additionally the 293t-cells were tested on their tolerace to TA-buffer in which the origamis are stored.
Since the binding between Fab-fragment and NIP is essential for coupling of the receotors, Alexa 488 linked origamis with and without NIP were given to the cells to detect the binding and the fluorescence was visualized with a LSM.
In order to test a simple transfection method for the 293t cells, Ca2+ precipitation with a lac z gene was done and the β-galactosidase was detected with an ONPG test. To test the functionality of the transfectionvector and the CMV-promotor in it, CFP and YFP was cloned behind the promotor and the plasmid was brought into the 293t cells with the same method.
