Collaboration - Team Heidelberg

The theme which the projects from team Heidelberg and our own share, is protein-tagging. We worked with 3 different tags to immobilize enzymes, team Heidelberg developed an intein-toolbox for protein-cyclization, protein-protein-coupling and other applications. When it comes to inteins (selfsplicing proteins), it’s quite useful to verify the sequence of the resulting protein. Knowing the original AA-code, that can be accomplished by mass spectrometry. Since we had access to a MALDI mass spectrometer, team Heidelberg sent us protein-samples in a piece of PAA gel. We extracted the Protein, digested it with trypsin and analyzed it. For the exact procedure of this MALDI analysis, please see the protocol. λ-lysozyme was tested in three varieties: once linear, and twice circular with different linkers. For the latter two, we were able to confirm that they were in fact cyclized in the predicted way, while the first was confirmed to be no linearized, just as expected.

Figure: MALDI mass spectrometry results of team Heidelberg's proteins

Additionally, in our conversations on protein expression, purification and testing, we brought up the difficulty we had overexpressing our fusion proteins and voiced our concerns that the modified pET-vector we used might be faulty. It just so happend that team Heidelberg had designed plasmid backbones this year, specifically for protein overexpression. They kindly sent us samples of their pSBX1K3 (BBa_K1362093) and pSBX4K5 (BBa_K1362097) backbones, so that we might test both high and low copy plasmids.

Furthermore, we contributed an article on our project for Team Heidelberg’s tumblr, where they post easy accessible articles on Synthetic Biology and molecular biology.