Team:Freiburg/Collaborations
iGEM is not only about the science, but also about meeting other iGEMers, helping each other out and meanwhile creating a network of scientists. In the context of our project, we collaborated with several teams, combining our projects and complementing each other’s knowledge in order to achieve our goals and cultivate the cooperative nature of the iGEM community.
iGEM Team Ruperto Carola
In July we visited the iGEM meetup in Düsseldorf to connect with other iGEM Teams in Germany. We met the teams from Heidelberg and Darmstadt and realized that our projects would be of great benefit for each other.
The project of the Ruperto Carola University in Heidelberg is based on receptors able to detect biomolecules such as peptides. We focused on creating these small peptides and therefore saw great advantages in using their receptor for the detection of our peptides.
We sent the team six peptides which we synthesized chemically via Solid Phase Peptide Synthesis in order to find fitting targets suitable for testing their receptors. We also planned an approach that involved lab experiments on both sides. This approach is based on the activation of the N-formyl peptide receptor 2 (FPR2) on the surface of leukocytes. Activation of the FPR2 caused by the toxin derived from Methicillin-Resistant Staphylococcus Aureus, PSMα3, and is detectable in a quantitative manner by measuring the calcium release. While we were using calcium release measurements with the calcium indicator Indo-1, this method was not perfectly sensitive, expensive and did not allow sample sizes as big as we needed.
Fig. 1: PSMα3 binds to FPR2 and leads to activation of the receptor, further causing calcium release that is measureable in a quantitative manner.
To tackle this problem, iGEM Ruperto Carola cloned chimeric receptors expressed on the surface of yeast cells. These receptors contain domains that originate from the native FPR2 receptor, as well as structural domains from the pheromone receptor Ste2, that ensure that the downstream signalling is still possible2. After receptor activation, the modified downstream signalling activates the transcription of a gene encoding a fluorescent protein. This gives a quantitative readout.
Fig. 2: Two chimeric receptors, one containing a yeast derived N and C-terminus of the Ste2 receptor, the other one only with a functional Ste2 C-tail, which makes downstream signalling possible.
During the cloning process, the downstream signalling activating the transcription of the fluorescence protein gene was mutated. We decided to use the system with a slightly different method: We detected the mating activity triggered by the Ste2-receptor22 activation by visualizing the diploid yeast cells formed by two different mating strains of opposite mating types, of which only one was tagged with a fluorescent protein. Using selection plates without leucine, we could detect diploid yeast cells, since they would be the only ones to express mCherry.
Fig. 3-6: From left to right: Confocal microscopy results of yeast cells grown on a plate containing 0,1 μM PSMα3. Confocal microscopy results of yeast cells incubated with 0,1 μM PSMα3 over night, DIC and mCherry. The samples on the not incubated with PSMα3 over night show slight fluorescence in comparison to those not incubated.
These results demonstrate the functionality of this system as a viable alternative to Indo-1 measurement, provided the receptor activation leads to direct fluorescent protein expression, giving a quantitative readout.
iGEM Team Darmstadt
The iGEM Darmstadt is working on a modular VLP based platform (MVP) that could possibly be used as a drug delivery system. Since they were still looking for a target to test this application, we offered an approach with cells in our cell culture in Freiburg. On our side, just as our collaboration with Heidelberg, the approach is based on detectable FPR2 receptor activation on leukocytes, which is caused by the toxin derived from MRSA, PSMα3. The receptor activation can be inhibited by a protein called Flipr, also derived from MRSA.
Fig. 7: GFP-tagged virus-like particles (green) were loaded with FLIPr using a sortase. FLIPr binds to FPR2 and spatial proximity is achieved.
The platform designed by TU Darmstadt is based on virus-like particles (VLP), which do not contain any DNA or RNA, but only coat- and scaffold-proteins. VLPs are synthesized in vivo and loaded ex vivo with the cargo of choice using an enzyme called sortase. Since the VLPs can be loaded with proteins, the smartest way to use them in our advantage was to load them with two peptides. On one hand, our inhibiting ligand against PSMα3, on the other hand a tag (FLIPr) that brings the platform near the place of interest.
The release of PSMα3 by Methicillin-resistentent Staphylococcus aureus (MRSA) recruits leukocytes, this is the major cause for an inflammatory reaction (REF). Therefore we decided to choose leukocytes as our target for the drug delivery system that the VLPs provide. To find a tag, we took advantage of a protein that binds to FPR2 receptors on the surface of these cells, called formyl peptide receptor-like 1 inhibitor (FLIPr) (REF).
At the beginning of the collaboration we did not have any inhibitory ligands yet. Therefore, we decided to load the VLP with FLIPr and, to facilitate visualisation, to tag the VLPs as well as the FPR2 with different fluorescent proteins (mCherry and sfGFP). Cloning and purification of the described VLP constructs, as well as the FPR2 transfection to HEK cells in the cell culture in Freiburg were succesful. As a next and final step, FLIPr mediated binding of the VLP to the FPR2 could be detected via flow cytometry.
Other Collaborations
iGEM Team Stuttgart
This year’s iGEM Team Stuttgart engineered a sustainable medium based on algae. In order to collect realistic growth curves, they sent their media to other iGEM teams to determine growth curve of a cell strain of choice in a different lab. To expand their dataset, we measured a growth curve for our Amber insertion optimized strain C321A.Δ in their three different media. The OD600 value was measured every 45 minutes over a time span of 7.5 hours. Subsequently, the three curves were plotted and sent to team Stuttgart.
Fig. 8: Growth curve of the bioincorporation optimized strain in the sustainable medium developed by iGEM Stuttgart
iGEM Team Dresden
We helped each other with mutual questions concerning cloning techniques. Further, we chose iGEM Dresden to be our first host for our mascot nietorP.
nietorP
Scientists are often overheard in our society. To prevent negative stigma, we as scientists not only have to stand for and promote what we discover, but also take the initiative ourselves. Specially climate protection is a topic that has been loudly vocalized by scientists. In order to contribute to action preventing climate change, we decided to give raise to the project nietorP. nietorP is our mascot, a giant Methicillin Resistant Staphylococcus Aureus. He traveled from Freiburg to Dresden to Berlin and even further and asked his host teams to use reusable cups, travel by bike, recycle or plant a tree, and thereby motivated other teams to join the movement. Hopefully we will see nietorP again in Boston, where he will tell us the adventures he experienced.
References:
[1] Kretschmer D. et al., Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus (2010), Cell Host & Microbe 7 (6), 463-73
[2] Merlini et al., The Royal Society Publishing (2013)
