Revision as of 21:01, 19 October 2016

Welcome to iGEM Aachen 2016

Collaborations

All teams consist of different people with different sets of skills. Therefore scientific collaborations are an excellent opportunity to exchange knowledge and are important to achieve a good project.

Click on the logo of a team to see further details about what they did for us or how we helped them.

iGEM Team Düsseldorf

iGEM Team Düsseldorf for iGEM Team Aachen

Our teams met for the first time at the German iGEM meetup in Marburg held in the beginning of August. There we exchanged our project ideas. We found out that both our projects are dealing with light activation. We discussed different ideas to irradiate our samples. iGEM Team Düsseldorf uses a so called “light box”, where the LED is fixed at the top and can be used to irradiate the samples with light of a specific wavelength. 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 a similar device for our purpose.

We used the device to study the activation of O-(2-Nitrobenzyl)-L-tyrosine (ONB-Y). ONB-Y was exposed to UV light for different exposure times. The activated ONB-Y samples were analyzed using gas chromatography coupled to mass spectrometry. The results reveal that when the exposure time was increased the cleavage of ONB-Y was higher. This was confirmed by the presence of a peak corresponding to 2-nitroso benzaldehyde i.e. the cleaved product.

Figure 1: Gladly receiving the "light box"

Figure 2: Handing over of the "Light-box"

If you want to see more details for what purpose we used the "light box", go to E.coli - targeting tyrosine irradiation point.
The counterpart of this collaboration can be seen on iGEM Düsseldorf's wiki.

iGEM Team FAU Erlangen

iGEM Team FAU Erlangen for iGEM Team Aachen

Our teams met for the first time at the German iGEM meetup in Marburg held in the beginning of August. The team from Erlangen consists of many chemistry students and so they are more into this field than we. Getting our non-canonical amino acids solved in various solvents turned out to be more difficult than we expected. So we asked them for help. They took the time to dig deeper into this topic and sent us literature they found. In addition, we arranged a skype meeting, where we discussed possible strategies for solving our non-canonical amino acids.

The counterpart of this collaboration can be seen on iGEM FAU Erlangen's wiki.

iGEM Team Technion Israel

iGEM Team Aachen for iGEM Team Technion Israel

The iGEM Team Technion uses a Tar receptor in their project fused to 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 team 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 using 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 3.

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 a test digestion and 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. In figure 4, colonies 2, 5, 7 and 15 showed bands with the right size. So we continued to work with these colonies to check for a fluorescence signal.

Figure 4: Gel of the finished expression system (different clones after ligation and transformation) cut with EcoRI and PstI; size of ~2800 bp for the expression system itself and ~2070 bp for the backbone pSB1C3 were expected. Clones 2, 5, 7 and 15 show a band with the right size on the agarose gel.

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 5 to 7. 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.

Figure 5: Settings of the Tecan Plate Reader for the measurements above and data of different samples of the expression system plus some controls: A (E. coli BL21 wild type); B (sample 2); C (sample 5); D (sample 7); E (sample 15); H (LB medium)Controls of E. coli wild type (A) and LB media (H) were taken to detect the background fluorescence in the media and the organisms we use. The data shows that all the expression systems, which we created (B-E), show higher levels of fluorescence than the controls. This indicates that GFP is expressed and our expression system works.

Figure 6: Data of the Tecan Plate Reader after subtraction of the background of the LB medium. Values for our control A (E. coli BL 21 Wildtype) and expression system (B-E) without the influence of the background noise of the LB medium.

Figure 7: Settings of the Tecan Plate Reader for the measurements above and data of different samples of the expression system and some controls. Incubation time was extended: A (E. coli BL21 wild type); B (sample 2); C (sample 5); D (sample 7); E (sample 15); F (LB medium).Controls of E. coli Wildtype (A) and LB medium (H) were taken to detect the background fluorescence in the medium and the organisms we use. The data shows that all the expression systems which we created (B-E), show higher values for fluorescence than the controls. This indicates that GFP is expressed and our expression system works.

After the successful detection of a fluorescence signal, we wanted to determine the position of the Tar receptor by studying the localizaion of the 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. 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 are functional. But there were also many colonies that did not have the fluorescence signal at the poles, which could be either caused by a misfolded Tar receptor or just due to statistical variation.

Figure 8: Image of colony 5 with fluorescence visible at the poles of some E. coli cells under the fluorescence microscope.

Figure 9: Closer image of E. coli cell of colony 5 with fluorescence visible at the poles of the cells. Image was taken with the help of the fluorescence microscope.

In our work we built an expression system for the iGEM Team Technion from Israel containing a Tar receptor-GFP fusion protein. In contrast to the iGEM Team in Israel we decided in agreement with their team 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.

We can confirm that a missing linker between the two proteins could cause a weak GFP signal or a misfolded 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 to localize in the membrane. 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 E. coli .

All in all, we showed that detection and correct 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 illustrates 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 work out as expected, they helped us to confirm 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 on how the modelling of our protein had 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 the 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 on how the folding process is influenced by our protease. He 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 sent us this document in the middle of September and because of this document we were able to assess 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.

The counterpart of this collaboration can be seen on iGEM Technion Israel's wiki.

iGEM Team TU Darmstadt

iGEM Team Aachen for iGEM Team TU Darmstadt

Our teams met for the first time at the German iGEM meetup in Marburg held in the beginning of August. As both our teams worked with the incorporation of non-canonical amino acids (ncAA) we exchanged our project ideas and experiences. From this basis a lively contact and a scientific collaboration evolved, that was of mutual benefit.
Here we describe our contribution to the exchange.
We are probing our DMNBS tRNA/synthetase variants with a fluorescence based, two plasmid screening system established by iGEM Team Austin Texas 2014. By using this system, fidelity and efficiency of ncAA incorporation can be determined. Though O-methyl-L-tyrosine (OMeY) is a well-known and frequently used tRNA/synthetase pair, it was not analysed before with the Austin screening system.

The OMeY tRNA/synthetase pair used by team Darmstadt was cloned into our pACYC derived backbone and transformed into E. coli BL21 cells, already containing the screening system. The cells underwent the same cultivation and screening routine as our DMNBS variants. For this, a microtiterplate was inoculated which was filled with medium of different OMeY concentrations (0-5 mM). The aim was to identify a sufficient concentration of ncAA to achieve high levels of fluorescence and growth rate to evaluate the incorporation efficiency.
Unfortunately, the measurement did not yield usable data, as the bacteria grew highly irregular and fluorescence signals were not comparable enough to evaluate.

iGEM Team TU Darmstadt for iGEM Team Aachen

A few team members of Team Darmstadt have knowledge of protein modeling. So they offered to simulate and evaluate the exchange of tyrosine with O-(2-nitrobenzyl)-L-tyrosine (ONBY) in the linker peptide. Thus we can value the effect the protection group of ONBY 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.

To elaborate the influence of ONBY on the structure of subtilisin E Team Darmstadt estimated parameters for the force field CHARMM27 in a similar way as they did it for ONBY. Afterwards they incorporated ONBY into the wildtype structure (PDB accession number: 3WHI). The structure was subsequently energy minimized using GROMACS 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 (root mean square deviation) over all frames in regard to the initial conformation was computed for both structures (figure 10).

Figure 10: 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 (figure 11).

Figure 11: 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 difference 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.

The counterpart of this collaboration can be seen on iGEM TU Darmstadt's wiki.