iGEM Team Duesseldorf

iGEM Team Duesseldorf for iGEM Team Aachen

Our teams met the first time at the German iGEM meetup in Marburg end of August. There we exchanged our project ideas. So we found out that our both projects are dealing with light activation. We discussed different ideas to irradiate our samples. Therefore, iGEM Team Düsseldorf uses a so called “light box”, where the LED is fixed at the top and you can shine light with a specific wavelength on your samples. This seemed to be a good device for our activation process, too.
iGEM Düsseldorf offered to build such a “light box” for our project as well. Because we need a specific wavelength for the activation of our non-canonical amino acid, we sent them one of our LEDs. They integrated this LED in their device and created so a device for our purpose.

iGEM Team FAU_Erlangen

iGEM Team FAU_Erlangen for iGEM Team Aachen

Our Teams met at the German iGEM meetup in the end of August for the first time. The Team from Erlangen had many students from the chemistry field, so they had better knowledge than us. Our problem of non-canonical amino acids (ncAAs) turned out to be more difficult than we expected. So we asked them for help. They went deep into the topic and provided us with literature they had found. In addition, we arranged a skype meeting, where we discussed possible strategies to solve our problem with ncAAs.

iGEM Team Technion_Israel

IGEM Team Aachen for iGEM Team Technion_Israel

The iGEM Team Technion uses a Tar receptor in their project fused with a linker and a GFP Protein. Based on this expression system the team wants to check the function and the location of the Tar Protein in the outer membrane of Escherichia coli.This receptor is required for chemotaxis in E. coli and used by the team in Israel for several purposes. Because of problems with the linker, our both teams suggested to build an expression system without linker to check the possibility of fusing the proteins without any linker sequence. Thus we are able to check its influence and can discuss, if the linker could be a reason for iGEM Team Technion’s problems.

To build this expression system, we used several parts from the iGEM Kit plate and put them together partly with the help of the common restriction sites of iGEM. For performing the experiment, we wanted to use the same conditions and parts, which the iGEM Team Technion used for their expression system. Therefore, we used the BioBricks BBa_E0040 (GFP), BBa_K777000 (tar receptor), BBa_J04500 (promoter and RBS) and BBa_B0015 (terminator) to build the expression system, which can be seen in figure 1.

The work for the collaboration started with the transformation of the single BioBricks. Afterwards, we cloned GFP and the Tar receptor as the first two parts of our expression system together by cutting BBa_K777000 (tar receptor) with SpeI and BBa_E0040 (GFP) with XbaI to form a scar between them and both with PstI to have them in one backbone. Then we integrated promoter and RBS via BBa_J04500 and accordingly BBa_B0015 (terminator) to our emerging expression system in the same way. We confirmed the successful integration of the composite expression system in pSB1C3 by performing gel-electrophoresis. On our agarose gel, we compared plasmids purified from 16 different colonies. We compared them with the control where the expression system was completely integrated and just the terminator was missing. On Figure 2. colonies 2, 5, 7 and 15 showed bands with the right size. So we continued to work with these colonies and checked for a fluorescence signal.

In conclusion, we checked the expression, the localization and the amount of the Tar-GFP Protein, produced by the created expression system. Therefore, we used the Tecan Plate Reader as device for measuring the GFP signal as you can see in figure 3 to 5. In these experiments we compared the background fluorescence signal of our wild type E. coli BL21 and the used LB medium with the positive colonies from the agarose gel. The evaluation shows that there is a huge difference of the fluorescence values between the wild type colonies (A) and the colonies of the produced expression system (2, 5, 7 and 15). This result indicated that our expression system works and GFP is successfully expressed through our expression system. We fulfilled the measurement with the device of the Tecan Plate Reader twice to verify our results of a fluorescence signal and expression of the Tar receptor- GFP fusion protein in our colonies. In the second measurement the incubation time of the cells was extended.

After the successful detection of a fluorescence signal, we wanted to determine the position of the Tar receptor with the combined GFP protein. Under the fluorescence microscope we compared one negative control of untransformed E. coli BL21 cells and the samples with the strongest fluorescence measured in the Tecan Plate Reader. The difference between these samples was also visible with this device and moreover, the fluorescence could be localized in the poles of the E. coli cells in some cases via the microscope. That means that our expression system worked and the proteins folded in the right way. But there were also many colonies that did not have the fluorescence signal at the poles, so the protein could not be folded correctly in every case.

In our work we built an expression system for the iGEM Team Technion from Israel containing a fused Tar receptor-GFP protein. In contrast to the iGEM Team in Israel we decided in agreement with their members to test the expression without putting any linker between the two components. Thereby, we wanted to test the influence of a linker and try the possibility of working without any linker.

So, we can confirm that a missing linker between the two proteins can cause a weak GFP signal and a wrong folded protein, which makes a localization of the Tar receptor impossible. In most of the cases the fluorescence signal was detectable in the cytosol, which indicates that the Tar-GFP protein did not fold in the right way. But we can also see that our expression system in general worked, because we were able to detect the fluorescence at the poles of some cells.

Because of our work the iGEM Team Technion knew that working without any linker could result in native folded proteins but is not the best option for secure results in their further project. The linker could still play an important role in the folding process of the Tar receptor and therefore influences the position of the Tar receptor in the membrane of Escherichia coli .

All in all, we showed that a detection and right location of the fused proteins could be possible even without linker. However, the rate of positively measured fluorescence in the requested/ desired location in the poles of E. coli was very low, which illustrate the importance of a linker between both proteins. With this work we could help the iGEM Team Technion finding the right focus of their problem by trying a different way of expression for them.


iGEM Team Technion_Israel for iGEM Team Aachen

The first approach of the iGEM Team Technion to help us was to model the incorporation of the non-canonical amino acid DMNB-serine in the active site of subtilisin E. As this did not worked out as expected, they helped us confirming the idea of theoretical inactivation by talking to several experts in their university. Based on the native structure of subtilisin E and the structure of the used non-canonical amino acid, they all agreed on the prosperity of that method. So we had an independent opinion about our project idea and were assured in following this theory once again. Moreover they gave us some useful hints how the modelling of our protein good have worked out and which parameters are needed to model the effects of the incoporation of our amino acid DMNB-serine.

The iGEM Team Technion did a lot of work when they tried to model our photocaged subtilisin E and to confirm the idea of inactivating our protease with the help of the unnatural amino acid DMNB-serine. In the beginning of their collaboration part the iGEM Team Technion met with Prof. Meytal Landau from the faculty of Biology at the Technion. She gave them some useful information and hints about visualization software and how a protein could be modelled with the help of this software. Afterwards they tried to draw our photocaged aminio acid with the help of the software Chemdraw. Unfortunately they were not able to draw our amino acid because of its complex structure. So the iGEM Team Technion contacted Einav Tayab-Fligelman with a request for assistance to draw our unnatural amino acid. In th next step the iGEM Team met with Dr. Fabian Glaser and asked him for assistance with the prediction of the final fold of the Subtilisin and it the folding process is influenced by our protease. This professor told the iGEM Team Technion, that it would be impossible to receive a credible result for the fold of our protease within the two months left in the iGEM project. Because of the time being the limiting factor, Dr. Glaser suggested to write a document which describes the steps for an exhaustive computational work, including some insights from the comparison of the structures. The iGEM Team Technion send us this document in the middle of September and because of this document we were able to the theoretical aspects of our project. Moreover members of the iGEM Team Technion created a 3D visualization of the Subtilisin and the photocaged serine. In the document they included some scientific remarks of Dr. Fabian Glaser. In this context we want to thank the iGEM Team Technion, Prof. Meytal Landau and Dr. Glaser for all their support and their help in this collaboration. The document of the iGEM Team Technion and all their conversations with different experts from their university gave us a chance to confirm the theoretical idea of our project and recognize the influences of our photocaged amino acid in the folding process of Subtilisin E.

iGEM Team TU_Darmstadt

iGEM Team Aachen for iGEM Team TU_Darmstadt

Our teams met the first time at the German iGEM meetup in Marburg end of August. There we exchanged our project ideas and found out that we are going in a similar direction. We are both working in Escherichia coli and with non-canonical amino acids. So we realised that this is a good basis for a collaboration.
In the following weeks, we organised some skype meetings. Thereby we described our ideas in a more detailed way and arranged our cooperation. We are testing our tRNA/synthetase pairs with a reporter plasmid, which Team Texas (2014) sent us.
By using this plasmid, you can evaluate if the tRNA/synthetase pair is working or not, based on a fluorescence signal. So we offered Team Darmstadt to test their tRNA/synthetase pair and their ncAA with this reporter plasmid as well.
This test shows directly if the ncAA is included. In addition, it is possible to generate data regarding the concentration of the ncAA for a most successful integration. The knowledge of these results is very important.

iGEM Team TU_Darmstadt for iGEM Team Aachen

A few team members of Team Darmstadt have knowledge of protein modelling. So they offered to simulate and evaluate the exchange of tyrosine with ONB-tyrosine in the linker peptide. Thus we can value the effect the protection group of ONB-tyrosine has on the structure of the protease.
This is pretty helpful because we can estimate if our approach can be successful at this site of the protein.