Team:TU Darmstadt/Collaborations

Collaborations with other Teams

Freiburg

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Since "The Real MVP" is a modular system for Virus-like particles (VLPs), it can be used for many different applications. One of them could be drug delivery. iGEM team Freiburg was interested in this application in particular and therefore we collaborated with them. They have developed an anti-toxin which should be used to bind the corresponding toxin and thereby neutralize it. To deliver the anti-toxin to the neutrophil cells, which should perform the described neutralization, they were interested to use our VLPs. As a proof of concept regarding drug delivery, the iGEM team Freiburg generated human embryonic kidney (HEK) cells with a receptor (FPR2) on their surface. We sent them our VLPs which were modified with a protein (FLIPr) that targets the FPR2. Team Freiburg wanted to perform an assay, where the modified VLPs target the FPR2 of these HEK cells.

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Introduction

We aimed for a modular VLP-based platform (MVP) that could be used for many applications. Since we decided on drug delivery as one of these potential applications, we collaborated with the iGEM Freiburg team. They developed a peptide via phage display that inhibits a toxin from S. aureus. According to iGEM Freiburg, neutrophil cells migrate to this toxin, as in a common inflammatory reaction and present a N-formyl peptide receptor 2 (FPR2) on the surface, which does not result in endocytosis  . We aimed to modify our VLPs with formyl peptide receptor-like 1 inhibitor (FLIPr) and the anti-toxin peptide from iGEM Freiburg. FLIPr targets formyl peptide receptor-like 1 (FPLR1) but at higher concentrations also the FPR2 of the neutrophil cells . Therefore, a drug delivery system is needed. This way the anti-toxin peptide could be delivered to the toxin and it could be inhibited as shown in Fig. 1.

Figure 1: Schematic overview of the targeting of the FPR2 on the surface of neutrophil cells by the VLPs modified with FLIPr.

The VLPs could be modified with FLIPr and the anti-toxin and would thereby target the FPR2 on the surface of the neutrophil cells. The neutrophil cells migrate to the toxin from S. aureus. After the binding of VLPs to the cells, the toxin is inhibited by the anti-toxin on the VLPs.

Schematic overview of the collaboration

As a first step of this drug delivery system, we wanted to modify our VLPs with FLIPr as shown in Fig. 2.

Figure 2: Schematic modification of the VLPs (sfGFP on the inside) with FLIPr via Sortase A5M.

Fig. 2 shows the modification of VLPs already loaded with Superfolder Green Fluorescent Protein (sfGFP). The VLPs have got a LPETGG-tag on their surface. The FLIPr proteins contain a polyG-tag. Due to these tags, sortase can modify the surface of the VLPs with FLIPr.
As a proof of concept, the iGEM Freiburg team generated human embryonic kidney (HEK) cells with FPR2 on their surface. They wanted to perform an assay, where the modified VLPs target the FPR2 of these HEK cells. This schematic procedure is shown below in Fig. 3.

Figure 3: Schematic procedure of the targeting of FPR2 on the surface of the HEK cells by FLIPr modified VLPs, loaded with sfGFP.

Fig. 3 shows the schematic procedure of the targeting of FPR2 on the surface of the HEK cells by our modified VLPs. The particles are modified at the surface with FLIPr and loaded with sfGFP to investigate the successful interaction via microscopy.
After the targeting assay, the HEK cells should be screened via fluorescence-activated cell sorting (FACS) and sorted into targeted and untargeted HEKs. This screening procedure is shown in Fig. 4 below.

Figure 4: Schematic procedure of the fluorescence-activated cell sorting (FACS) of the VLP targeted and untargeted HEK cells, FSC= Forward Scatter, SSC= Side Scatter.

Fig. 4 shows the schematic procedure of the FACS, how the VLP targeted and untargeted HEK cells would be differentiated. The HEK cells are aligned so that ideally, they flow through the laser beam one cell at a time. The scattered light is characteristic to the cells and their components. The HEK cells are labelled with the fluorescent VLPs so that light is absorbed at first and then emitted in a band of wavelengths. The detector system converts the measurements of forward-scattered light (FSC) and side-scattered light (SSC) as well as specific fluorescence signals into digital signals that are processed by a computer. Afterwards, the HEK cells are sorted into targeted and untargeted cells to identify the targeted population and proove the drug delivery with the modified VLPs.

As a first step of the collaboration with the iGEM team Freiburg, we succesfully cloned mbp-flipr via gibson assembly into pACYCT2 backbone. Afterwards, we verified the success of our cloning via sequencing of the generated plasmid. With a not mutated plasmid of mbp-flipr we transformed E. coli Top10 and BL21 (DE3) cells and generated glycerol stocks. The iGEM team Freiburg transfected their HEK cells with FPR2. Due to time issues it was not possible to generated any further data. The next step to achieve a targeted drug delivery would be a successful purification of MBP-FLIPr and a proof of the same with a SDS-PAGE. Afterwards our VLPs would have to be modified with FLIPr and the iGEM team Freiburg would have to verify the drug delivery by FACS.

Bielefeld

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The iGEM team of Bielefeld contacted us for a possible collaboration regarding the standardization of fluorescence data which is generated under the most varied conditions. Standardization of GFP measurements was achieved during the InterLab study of the last years. This year, Bielefeld wanted to establish a standard for mCherry. Therefore, Texas Red was chosen as a reference.
As we planned on using mCherry in our project anyways, we considered this collaboration as fitting. We used Texas Red, which team Bielefeld kindly provided us with, to create a dilution series. We also took a measurement of mCherry, which we planned on using to modify our VLPs via the sortase reaction at first, to then be standardized using the Texas Red data generated by us and probably many other teams. Thanks to team Bielefeld for organizing the standardization of a fluorescent protein other than GFP.

Duesseldorf

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For several years now we have designed postcards and sent them to Duesseldorf. Every year their team collects numerous postcards from many iGEM teams around the world and then send a postcard of each team back to everyone who participates. Each time it brings a lot of fun to see what designs the other teams have come up with.
Thank you Duesseldorf for keeping up this collaboration every year. We can only imagine how time consuming the organization of this collaboration is. But it is always a great opportunity to learn something about the other projects and to get in contact with teams from all around the world. Keep up the tradition for years to come, as again it was a pleasure for us.

Marburg

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iGEM Marburg tried to make all of our lives easier this year. They worked on a colony picking bot on the basis of the Opentrons OT-2. In order to achieve their goal, they needed as much help from the iGEM community as possible.
Therefore, they were asking other teams to send pictures of agar plates with colonies on them, which they needed to train the artificial intelligence. We gave our best to send iGEM Marburg as many pictures as possible and we even sent in the second most pictures from all participating teams. Hope we could help you, iGEM Marburg.

Stuttgart

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iGEM Team Stuttgart contacted us for a collaboration. The aim was the establishment of a medium based on algae. This medium was supposed to be more sustainable and used for cultivation of bacteria instead of conventional media.
We received three different kinds of media and tested them by generating growth curves. For recording of the growth curves, we used E. coli BL21 (DE3). Every 45 minutes over a time span of 7.5 hours the OD₆₀₀ was measured. Subsequently, the three curves were plotted and sent to team Stuttgart. They collected the results from all participating teams to make sense of all the generated data. We are excited to see whether any of the provided media will replace conventional media in the future.

Meetups

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2019 seemed to be the year of iGEM Meetups. iGEM TU Darmstadt participated in iGEM Spring Festival (Bonn, May), iGEM meetup for teams and supervisor (The Hague, June), German iGEM Meetup (Duesseldorf, July) and InParis European Meetup (Paris, July). The meetups provided some good opportunities to exchange ideas, get to know other teams and their projects, find collaborating partners during numerous poster sessions and also enjoy expert talks.
Besides getting in contact with other teams, the meetups always are a great opportunity to obtain feedback from other scientists and to prepare for the presentation and the poster sessions in Boston at the end of the year. Meetups are also useful to receive suggestions for improvement of the project. Therefore, we would like to thank iGEM team Bonn, Rathenau and RIVM, iGEM team Duesseldorf and Pasteur Paris, Ionis Paris, GO Paris Saclay and Evry Paris Saclay iGEM teams for the great organization of those events. We really enjoyed to participate and to get to know many of the other iGEM teams from around Europe.

Collaborations outside of the iGEM community

Lab³

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The open community Lab³ is a non-profit organization which was founded in 2015 and is located in Darmstadt. The vision of the community is to build up open laboratories and workshops, where everyone can realize their own research in the fields of electronics, engineering and life sciences. Currently, the community consists of an electronical laboratory, a workshop room and a creative laboratory. The laboratories are equipped with soldering tools, a cnc machine, a laser cutter, several 3D-printers and personal computers with CAD (computer aided design) and CAM (computer aided manufacturing) software. This year, our iGEM team engaged a collaboration to develop parts for the bioreactor and also set up a server cluster from the scratch for our modeling.

Servercluster

For our computational expensive modeling we were looking for a way to cover the time necessary for computing at an early stage. Together with the Lab³ we worked on setting up a sever cluster (see Fig. 1 and Fig. 2). For the construction of the server cluster we decided to use seven Supermicro nodes with 24 cores and 64 GB RAM each. These nodes where connected with a network switch to a master server which manages the infrastructure. On the server cluster we ran the Linux distribution Ubuntu with the modeling tools Rosetta, GROMACS and TensorFlow. To accelerate our calculations in the fields of machine learning and molecular dynamics, we used a special server with Nvidia GTX 760 graphic cards.
Machine learning approaches are very common today. We used this technology for the 3D-Structure prediction of our Sortase A7M. In the project the software tool GROMACS was used to validate the stability of the computed sortases. You can see the results on the Modeling Page.

Figure 1: Node servers with Dell GPU server.

Figure 2: Backplane view of our nodes and network connection.

Labware

The 3D-printing room of the Lab³ was a nice playground to learn a lot about printing technologies. We designed several parts for the bioreactor and printed them with fused deposition modeling (FDM) printer Prusa MK3. In this very popular printing technology, a thermoplastic filament gets molten in a hotend and the liquefied material gets build up on a platform, layer per layer. With this technique we were also able to create some new wide combs for the agarose gel extraction, which we needed in our wetlab (see Fig. 3). Aside this technology we also printed the combs and other stuff with a Multi Jet Modeling printer from Stratasys. This printer works with a photopolymer, which makes the printer very precise. In Fig. 3 you can see a printed comb with the Multi Jet Modeling printer. In order to support our public engagement we designed a 3D-model of a Virus-like particle (VLP) (see Fig. 4). With this we were able to visualize the structure of VLPs and explain our idea of a modular platform.

Figure 3: Comb printed with Multi Jet Modeling printer.

Figure 4: VLP with modifications, printed with a FDM printer.

Tech parts

Over the summer, the team members involved with the Tech project part was able to work in the laboratories of the Lab³ community. By means of the lab's tools we built up the OD₆₀₀-sensor. The syringe pumps meant for induction were printed with the FDM 3D-printers. We also used the solder stations to manufacture printed circuit boards (PCBs) for the control of the SensorBricks. The bricks where manufactured with a milling machine and a drill.
See more at the Tech page.

Figure 5: Prusa FDM printers.

Paul-Ehrlich-Institut (PEI)

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While working on our project, we talked to several experts. One of them was Dr. Stefan Schülke who works at Paul-Ehrlich-Institut (PEI) in Langen, Germany. PEI is a federal institute which works on the approval of different drugs (e.g.: vaccines, allergens for therapy, drugs for gene therapy, and many more).
Dr. Schülke is part of a group which focuses on the research field of molecular allergology. While talking to Dr. Schülke, who also teaches at TU Darmstadt, he offered that we could analyze our product at the PEI. He also worked with Virus-like particles (VLPs) before and therefore was able to give us some valuable input for the project concerning the immunogenicity of our Modular Virus-like particles (MVPs). Together with a former iGEM TU Darmstadt member, Alexandra Goretzki, we performed the experiments. You can read more about this here.

References