Collaboration with iGEM Göttingen

The University of Göttingen helped us testing the cytotoxic activity of miniColicin in other organisms than E. coli. Shimwellia blattae was transformed with miniColicin without mutation (\(\text{mCol}_{\text{WT}}\)) and with mutation (C266A) \(\text{mCol}_{\text{Mut}}\) under control of the T7 promoter (BioBrick BBa_K525998) on the BioBrick backbone pSB1A3. We sent Patrick (Team Leader of the killswitch team) to Göttingen and he was in their lab to carry out all the experiments. It was a great experience to work in another lab and we learned a lot about the Göttingen iGEMteam and how they are structured. Of course, there were also some social events which leads to us and iGEM Göttingen stay in contact and visiting each other. Thank you for this great exchange!
The Shimwellia blattae cells were transformed via electroporation with the miniColicin variants and cultivated on LB-Agar plates supplied with ampicillin. This procedure was performed twice. In order to ensure that pSB1A3 is suitable for amplification in Shimwellia blattae, original pSB1A3 was also tested.
No significant difference in colony amount on each LB-Agar plate was obseverd between the two Minicolicin constructs: \(\text{mCol}_{\text{WT}}\) transformed cells yielded \(220\pm20\) colonies and \(\text{mCol}_{\text{Mut}}\) cells yielded \(200\pm50\) colonies (mean of the dublicate with standard deviation).

Collaboration with iGEM Aachen

Since our iGEM team as well as the team of the RWTH Aachen worked with an expanded genetic code via amber supression, we exchanged our knowledge on this topic. Furthermore they tested the incorporation efficiency of our orthogonal tRNA OMT-RS pair with the "Expanded Genetic Code Measurement Kit". In exchange we modeled the molecular dynamics of Subtilisin E with the nnAA O-(2-nitrobenzyl)-l-tyrosine (ONBY) at the specified position.

To elaborate the influence of ONBY on the structure of Subtilisin E we estimated paramteres for the force filed CHARMm27 in a similar way as we estimated parameters for O-methyl-l-tyrosine. Afterwards we incorporated ONBY into the wildtype structure (PDB accession number: 3WHI). The structure was subsequently energy minimized using GROMACSS 5.0.3 in TIP3P water and after equilibration simulated over 50 ns resulting in 5001 conformations. We applied similar conditions to the wildtype protein.
The RMSD over all frames in regard to the initial conformation was computed for both structures and is displayed in figure 1.

Figure 1:RMSD of Subtilisin E wildtype (red) and the mutation variant (blue) in regard to the initial conformation.

The conformation with the smallest RMSD towards the consensus structure was chosen to represent the overall conformation and is displayed in figure 2.

Figure 2:Overlay of Subtilisin E wildtype (red) and mutant variant (yellow). The mutated amino acid sidechain is displayed as blocks in PyMOL.

It is evident that there is no relevant fdifference between the wildtype structure and the mutant variant. Therefore we can conclude that the incorporation of ONBY at tyrosine 77 is not sufficient to alter the structure. This can be traced to the fact that ONBY is introduced at a surface position.

Collaboration with TU Munich and LMU Munich

One part of our robot is a do-it-yourself syringe pump which we constructed and 3D printed on our own. Also the iGEM team of Munich had the idea to work with syringe pumps. They want to construct a feature to easily upgrade a 3D printer to a bio printer. The essential part for this transformation is a syringe pump, they constructed. Since our robot basis is a 3D printer and we also use a syringe pump it was natural to work together. Munich sent us their system and we printed the additional parts on our own. This is exactly how they imagine their system. We tested it and gave them feedback on their design and possible challenges, so they can improve their design. For us it was useful to test the multi purpose of our robot and how easy it is to add new parts and features. Also the syringe pump design is different and we had a good chance to evaluate our design and compare it to the one of Munich. We derived useful information within this collaboration and are interested in working closer together in the technical part of the iGEM project. Especially if Munich decides to go on focusing on the do-it-yourself sector, Darmstadt is related to. We like to thank the iGEM team of Munich for this opportunity and wish them success with their project. Keep on working :)