Attribution

Attribution is an important point of any iGEM project as it is necessary to set the results a team obtained into the context of the support they obtained. Thus, we'll try to describe honestly how our project evolved, which student was responsible for which sub-project and who supported our project with a scientific or non-scientific contribution.

Finding the biotINK project

For the history about how we found our project it is somehow important to know that there was no iGEM team from TUM or LMU in 2015 and no team in 2014 at TUM. Thus there were in each year students that wanted to participate in the iGEM competition but there was no hosting lab that offered the support necessary to make the "iGEM experience". Thus there has been a long time in which students have brainstormed to find some interesting ideas that they could convert into an iGEM project. Also our bioprinting project is far older than this year, at least in theory. The life science students finally found together approximetely in February at at this time point they had already few promising iGEM projects in mind. The project ideas that were developed the most were i) transforming of the planet mars with bacteria, ii) creating a vegan substitution product for milk and iii) bioprinting. Initially the bioprinting project was seen as the most interesting project-idea. Unfortunately, there has been a really nice project on bioprinting with bacteria by the TU Delft iGEM team in the last year and thus the team has discussed a long time if it is a good idea to participate with another bioprinting project. In the end we agreed that that there will most probably a bioprinting track will be established in the next years and that this topic has very high potential. By the way was it really encouraging when we listened to Randy in Paris when he described the potential of SynBio and used bioprinting of organs as his example of choice! At this point we were totally confident that our project choice has been totally right.

Our prinicpal investigators (PIs)

As I told you in the last paragraph it has been very difficult to convince a professor at TUM and also at LMU it was difficult to find a PI that is enthusiastic about iGEM after Prof. Mascher has left Munich. The initial spark for the foundation of the iGEM team was given by the Graduate Center GRK 2062: Molecular principles of synthetic biology that wanted to reestablish an iGEM team in Munich.

Prof. Dr. Jung (one of our secondary PIs, LMU) is the head of this graduate center and thus initially started the team by recruiting students. But she did not want to hast the project fully at her lab and so she (and the GRK) looked for a lab participating in the graduate center that could host the iGEM team as a main lab. In the end we got a small lab space in her lab in which we could do some cloning work as the distance between LMU and TUM at Weihenstephan campus is more than 2 h with public transportation. We are grateful that Prof. offered us labspace but in the end we decided to do all work in Weihenstephan as it turned out to be difficult to separate a project into two labs. Prof. Jung was not at all involved into the science of the iGEM project.
Prof. Dr. Skerra (our main PI, TUM) has already been the main PI of the iGEM teams from TU Munich in 2012 and 2013 and already had some experiences of how it can be to host an iGEM team. In the beginning he did not want to host another iGEM team at his lab but in the end we could convince him that he participates another time in iGEM. He is a well known expert on the field of protein desing, especially for therapeutic proteins and also foundet two successful companies. For this reason he is quite busy and could not invest too much time for the iGEM competition. In this year he joined our weekly team meeting only once, when we had decided on our project. In this session he gave us several very valuable hints, what components we could integrate and what aspects we should focus on in order to get a proof-of-principle in the end. Besides this discussion session with him it is his philosophy that iGEM is a students competition and that the students should design and create the project. He generously offered everything of the resources available at his chair together with the excellent analytical equipment but let us the freedom to plan the project on our own - and we are really grateful that he gave us this opporthunity!
Prof. Dr. Westmeyer (secondary PI, TUM, Helmholtz center) was asked to join the team in the middle of the project when we realized that we needed some expertise in the field of fluorescence microscopic imaging. He supported us especially in the last weeks with cellbiological reagents for stainings and transfection and with the analytical instrumentation of his lab. As he has spent the last years in Boston at the MIT and at Harvard he is very openmindet concerning the potential and possible applications of SynBio in the future.

Our Journey Through Munich

During the course of our project we have met many nice people willing to help and support us with lab space, knowledge or materials. Below you can find all the stations we took throughout our journey to the final result of our project.

Attribution sorted by methods

All work associated with planing, conducting and the evaluation of experiments was done by student-members of our team. Instructors have assisted the students in the planing of their experiments, have introduced molecularbiological and chemical techniques to the students when they performed a specific experiment for the first time. Also the evaluation of the experiments was done by the students (after an introduction to the theory and some hints what to do and what to avoid). After a preliminary evaluation of the results, data were discussed in the weekly team meeting where our two instructors Andreas and Volker (both PhD students at the lab of Prof. Skerra) were present to reflect our project. Here we mention for every technique performed during our project to allow the judged to directly contact the student that has performed the specific task they are interested in.

Protein Production and Purification

As we produced, purified, conjugated an analyzed many different protein samples different team members were involved, including Niklas, Jan and Julian. Shaker flask productions were done totally independent of the lab members, for fermentation (which turned out to be a bit more difficult than explained in lectures) one of our Instuctors had a look on the whole production. Purification with normal columns was done alone, as soon as a Äkta-purifier was involved we had to have someone from the lab who had a look on our handling for the first 3 to 5 chromatographies.

Molecular Cloning

In the first week we had an introduction to molecular cloning, afterwards we worked independently from our Instructors and only asked them from time to time if some problem occured. The gene design for all BioBricks we made by a student member (often Clemens or Christoph) and was then reviewed by our Instructor Volker.

Western Blot

All Western Blots were done by Luisa and she got an intoduction to blotting and staining from Fabian Rodewald and Christian Deuschle.

Mass spectrometric measurements

The coretness of recombinant proteins before and after conjugation was analyzed by ESI-TOF mass spectrometry. Sample preparation was done by student members of the team, the actual measurement was done by a technical asistant (but also the PhD student do not have to measure themselves).

Flow Cytometry

The flow cytometry experiments were all performed by Luisa as she has spent 18 month in a research group in Hamburg before she started her studies in Munich. Thus she already brought all the skills to measure all our cells in FACS.

Fluorescence Microscopy

The fluorescence microscopy at the lab of Prof. Skerra was performed by various different people as it was necessary for many different sub-projects. The high resolution confocal fluorescence microscopy was done at the Helmholtz Center in Munich in the Lab of Prof. Westmeyer by Christoph as he will also do his master's thesis in this lab. He got the introduction into staining cells and performing confocal imaging experiments from our external Instructor Jeff.

Scanning Electron Microscopy

Our colorful Scanning Electron Microscopy images were done by Prof. Dr. Wanner from the Plant Science Department of the Ludwig-Maximilians University. Elena approached him to support our project in terms of a collaboration providing us some impressive electron microscopy images of cells. Prof. Wanner facilitated all required reagents for the sample preparation such as microscopic slides, glutaraldehyde, transport boxes, ...! The sample preparation was performed by Clemens late in the evening, thus being a bit improvised. The samples were then transported and picked up by Jan and Elena, bringing them to the SEM facility. Afterwards, the remaining sample preparation steps after the fixation were carried out by Prof. Wanner and his coworkers. We are sincerely grateful for his support!

Blood samples

For our "linker chemistry" subproject we needed fresh blood samples that contained human biotinidase. For this purpose Vivien, the student hat performed all the chemistry experiments, went to PD Dr. med. Thomas Skurk who helped us with by taking blood samples.

Chemical synthesis of the Biotin-NHS compounds

Chemical synthesis was done by Vivien as she has already done here Bachelor's thesis at the Chair for Organic Chemistry at TUM. The initial synthesis plan was discussed with Dr. Martin Dauner, who is the chemist in the lab of Prof. Skerra. Vivien worked alone after during the preparation of the compound and the subsequent analysis of their properties. The NMR measurement were then measured at the chemical faculty in Garching. The evaluation of all results was done by Vivien (including NMR evalution).

Maya Video

To be honest we are very proud that the Maya Video Vivien made looks so great in the end. In the beginning the first renderings she showed were fare more simple than what it looks now. She has already had some experiences in the field as she has done graphical design with blender as a kind of hobby before and this helped her to understand the Maya software faster (which is really not that intuitive). She also put up a How-To Video in which she explains the creation of the video in app. on hour. If you are interested in the video, please ask her as she really did it on her own ;)

Micromanipulator experiments at the Chair of Livestock Biotechnology of Professor Dr. Schnieke

Figure 1: Javier operating the micromanipulator

We have to thank Professor Dr. Schnieke and her [http://btn.wzw.tum.de/ Chair of Livestock Biotechnology] at the WZW (TUM School of Life Sciences Weihenstephan) for so generously providing us with the possibility to use the micromanipulator at the chair's laboratory and allowing us to conduct some very specific experiments with our bioink. With the extensive help and instructions of Dr. Nicolas Ortega, researcher at the lab with a lot of experience with the device, we were able to plan experiments and test our bioprinting technique on a very tiny scale. Thereby our team members Julian and Javier were able to observe the ejection process under controlled conditions in great detail and document the behavior of our modified HEK cells containing ink. Thanks to this opportunity we could obtain useful and valuable visual material as well as a better understanding of our biotINK concept.

Business plan

The Businessplan was done by Christoph as he also sudies TUM-Economics besides his studies in Molecular Biotechnology.

Survey: "Supporting Entrepreneurship"

For the creation of this survey we had a special Instructor, Philipp. He is a PhD student an the Chair of Technology- and Innovationmanagement. He supervised Hallie who is a US-american exchange student who joined our team to create this survey and find out how we can make iGEM more productive concerning it's output in successful start-up companies.

Team:LMU-TUM Munich/Attributions