This iGEM year we once again had an amazing experience while creating our own project in the field of synthetic biology. We were able to intensively research our project, collect our funding and study our parts both in the dry lab and wet lab. Our project PHIRE BYRD provides a realistic proof of concept for the implementation of an adaptive immune like system to engineered biofilms for a multitude of possible applications.
But we did not do this alone! Without the support of many great and helpful people we would not be where we currently are. Many experts in various fields of studies supported us throughout our journey and significantly contributed to this project.
We do owe a lot to them, so we would like to mention them here and use this opportunity to thank these lovely people!
Like last year, we were affected by the restrictions of the COVID-19 pandemic. Therefore, only a far-reaching theoretical elaboration of our project seemed feasible at the beginning of our project. Especially these circumstances made the feedback and advice from external experts more valuable than ever before. Nevertheless, we did not gave up hope and were even able to organize a few weeks in the lab with a lot of effort, based on a hygiene concept in the context of infection control measures. Despite all the obstacles we had to overcome, we managed to stick together as a team and build a project we are very proud of.
First of all, we want to thank our primary PI Prof. Dr. Heribert Warzecha for enabling an iGEM Team at our university. Because of his incredible commitment to this project, we all had the amazing opportunity to be part of iGEM and enjoy this inspiring journey. Despite his responsibilities as group leader of his lab and vice president of the TU Darmstadt, he was always there to support us with our project and help us with different challenges we faced. We also want to thank our secondary PI Prof. Dr. Dominik Niopek for his dedication towards our project and his continuous effort to discuss our project. He also helped us a lot with the theoretical elaboration of our project. Both PIs were extremely supportive in setting up our experiments and a hygiene concept for our lab phase. Without their help we would not have been able to realize our theoretical concepts short term in the wet lab. During this intensive time Simone Bartl-Zimmermann always had our back, helped us to organize our materials and took care of all orders that had to be made. She always took the time to help us with our questions with a lot of patience. We additionally would like to thank Barbara Wolf for the help regarding the organization of our sponsoring and communicate with various university offices. Also, we would like to show our gratitude to Dr. Melanie Mikosch-Wersching for supporting us with our correspondence to sponsors and our public appearance. Throughout the whole iGEM year, the project advisors have been a particularly important constant for our project. Starting with our initial project choice, the project elaboration, continuing with the proofreading for the wiki and extensive help in the lab they contributed with great scientific and social advice. It was their knowledge and experience that helped us to guide our ideas and concepts to a well elaborated scientific project. We would like to thank Benedikt Spannenkrebs, Christian Sürder, Giulia Ravagnan, Jan Kalkowski, Leon Kraus, Maximilian Zander, Sebastian Barthel and Yannick Kristiansen for this.
TU Darmstadt
We want to thank Dr. Guido Klees, Dr. Kirsten Santelmann and Kirsten Wehner from the Lernlabor Biologie at our faculty for letting us use their laboratory spaces! We were very happy to get the possibility of performing experiments in the lab.
We would like to thank Dr. Alexander Rapp for instructing and providing us with the fluorescence microscope from the Cardoso Lab. He was a tremendous help in understanding our own fluorescence microscope and contributed to the amazing fluorescence images of our biofilm.
Dr. Wadim Weber extensively explained the working mechanism of holins to us and proposed some possible systems that could eventually work on our concept. He also proposed methods for the optimization of the pore size and the amount of the produced holins, which therefore helped us to make the phage release concept efficient. In the wet lab he was a great help with the troubleshooting of our issues with BL21(DE3) IPTG induction. He also discussed our genetic constructs with us, addressing possible issues with the spacer sequence between RBS and the corresponding start codon. Besides him we want to thank Philipp Kemp, M. Sc. from the working group of Prof. Dr. Victor Stein who has helped us here as well. They have been a great support when we were working in the lab.
For our experimental planning, discussions about our results and advice in the lab, Jan Mathony, M. Sc. was a great help. We were always able to ask questions and get his feedback, which helped us to efficiently use our time in the lab. Also, we want to thank Sabine Aschenbrenner for helping us in the lab as well.
Dr. Carlo Klein helped us to plan our assembly strategies by Golden Gate Assembly for our parts and genetic circuits. Additionally, he provided us parts for a Golden Gate Assembly test run in our lab. This support was extremely useful during the planning of our wet lab phase and within the first two weeks in our lab.
Experts
Especially during the research phase of our project, specific feedback on the researched topics from experts in these fields was extremely valuable. Our in-depth research was proved to be very difficult without contact to local stakeholders or correspondence to the authors of the papers we read.
Also, for the validation of our theoretically planned genetic circuits we received a lot of help from experts. Our lab phase could not have been that efficient without the feedback we got from them! Their enthusiasm and willingness to help fueled our project and provided a huge motivation. Therefore, we would like to thank each expert for their contribution to our project.
Prof. Dr. Anke Becker is a professor of microbiology and PI as well as the director at the center for Synthetic Microbiology at the Philipps-Universität Marburg in Germany. She kindly answered our questions concerning the possible difficulties of our quorum sensing molecule (AHL) passing through the Gram-positive cell wall of B. subtilis. Additionally, she suggested an experimental design with which we could test her hypothesis. Prof. Dr. Fabian Commichau shared his knowledge of AHL with us, upon which we could draw further conclusions concerning our theoretical lab plan as well as possible applications for our project. We based our genetic circuit to detect Pseudomonas aeruginosa on the genetic circuit presented in one of Ms. Ying Wu publications. She kindly gave us access to two plasmid sequences used in her genetic circuit.
We appreciate Dr. Anna Dragoš for suggesting the idea of integrating the lambda attB site from E. coli into B. subtilis so that it is also compatible with lambda phages. Dr. Jason Gill talked to us about the phage integration site helping us to identify possible problems and discussing suitable alternatives. Zihao Yu discussed our output design with us and confirmed our approach of using a single gene as a direct output of our switch. We especially would like to thank Prof. Dr. Grzegorz Węgrzyn. He helped us a lot to make our genetic circuit more efficient by recommending us the RecA730 mutant. He provided a lot of help during the optimization of the genetic circuit for RecA730 and gave us a lot of valuable feedback on our faculty presentation. We would like to emphasize that Prof. Węgrzyn not only gave us valuable input but was also so generous to provide us with lysogens, bacteriophages and related protocols.
Dr. Ilka Bischofs was the first expert we talked to about biofilms. Firstly, she advised against working with the strain 168 as it has been domesticated and is therefore not able to form a biofilm. Furthermore, she referred us to Prof. Dr. Ákos T. Kovács, a B. subtilis biofilm expert from the Technical University of Denmark (DTU). Based on Prof. Dr. Kovács recommendations and advice we planned to cultivate both a cheater system as well as a co-culture of two differently genetically modified B. subtilis cells from the same strain NCIB3610/DK4210. We are especially grateful to Mr. Kovács since he provided us with plasmid maps and some guiding paper recommendations. Additionally, he sent us fluorescence marked B. subtilis strains and transformed E. coli strains boosting our theoretical and practical part significantly. Professor Dr. Daniel Kearns from the Indiana University in Bloomington, USA is an expert for the domestication and biofilm formation of B. subtilis. His input helped us finalizing our lab plan and highlighted potential mistakes that needed some adaption.
Prof. Dr. Beat Lutz is an expert for Cre recombinase induced inversion of genetic elements with lox66/lox71 sites. He emphasized the importance of the correct orientation of our lox sites and corrected a sequence error within our switch. Prof. Dr. Jan-Willem Veening gave us advice regarding codon optimization. He was also a big help with the choice of a suitable terminator for B. subtilis recommending the rrnB terminator. Dr. Thomas Gorochowski helped us design a logic gate based on our kill-switch and even briefed us on designing an assay concept.
The correct communication of science to society and ethical aspects in modern biosciences are more relevant than ever with the development of potent tools for genetic engineering. Therefore, our team really focused on bringing our science to people that are either directly or indirectly influenced by it. Also, we elaborated the possible risks, hazards and restrictions evolving from our project with our ethics and policies team. We received a lot of input from experts in these fields and are very grateful for their help. Dr. Michael Vockenhuber talked to us about the importance of regulations in relation to the release of GMOs. He is the Biological Safety Officer at the Technical University of Darmstadt and thus is very experienced in this area. The conversation with him encouraged us in our efforts to implement a kill-switch. Talking with Dr. Johannes Fritsch led us to conduct a survey on the acceptance of GMOs in society. He also referred us to LAGeSo and ZKBS. We talked with Prof. Dr. Alfred Nordmann about the importance of meaningful and reliable sources, especially in the context of synthetic biology. Based on this, we decided to integrate the Responsibility, Research and Innovation (RRI) concept into our Human Practices. Dr. Swantje Straßheim pointed out the failures of science communication on the part of synthetic biology and how important it actually is, which encouraged us to focus even more on science communication in our project. Prof. Dr. Kathryn Nixdorf is a founding member of IANUS. She reviewed our project for intrinsic biosecurity risks and confirmed our intention to integrate a kill-switch. We also spoke with an employee of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety, whose identity must remain protected. The staff member explained to us how important it is to carefully weigh the benefits and risks of GMOs.
Former iGEMers
iGEM has a long tradition at the TU Darmstadt. Fortunately, there are always former participants who have kept their curiosity about the following years and are willing to support with their knowledge and experience. They often helped with organization matters, took part in team meetings from time to time, gave feedback on meetings and provided workshops for the new team. We would like to thank Bea Marie Spiekermann, Leon Werner, Rosi Krebs and Timotheus Martin Kiehl, who did a presentation training with us to prepare us for our faculty presentation and the final presentation.
Especially with our modeling we received a lot of support from our predecessors. We would like to thank Fran Bacic Toplec and Tom Müller for their helpful Python seminars. We are especially grateful to Tom Müller who not only regularly participated in advisory meetings but also held a Clean Coding seminar, a Biotite seminar and joined regularly for our meetings on the Phoenix project. Jonathan Funk and Ben Mayer also supported us and played an important role in the development of our software concept.
Proofreading is indispensably important to create a successful wiki. For this purpose, a large number of texts and images have to be checked several times. We would like to thank Mehryad Mataei, Tom Müller, Robert Klein, Angela Kühn, Bea Marie Spiekermann, Jan-Philipp Kahl, Jillian Hoffmann, Rosi Krebs, Johanna Möller, Tizian Dege, and Vincent Gunawan.
Others
A lot of supporters of our project do not fit in the above-mentioned categories but also contributed significantly. We would like to thank them for their time and advice, which really pushed our project and allowed us to have this amazing iGEM experience. We are very grateful to Julian Benz, who is a teacher at the Friedrich Ebert School in Pfungstadt. He helped us with experimental material for the school workshop, which was held for his biology class and gave us valuable feedback. Stephanie Schultheis is a social worker in child and youth welfare. She helped us create our stories for the Tonie-Boxes in a child-friendly way by looking over our finished stories. Sabine Schiner is the Darmstadt & Süd Hessen editor of the Darmstädter Echo. She wrote an article about our project, helping us to connect with society. Likewise, we like to thank Petra Gahabka, editor at TIP-publishing, for writing about our kindergarden visit. We would also like to thank Dr. Sascha Vogel, founder of the Science Birds GmbH. He held an awesome science communication workshop for our team and was a great enrichment for all of us.
We also want to thank a lot all the people that supported our Greener Lab Initiative. We appreciate the help of Prof. Dr. Nico Bruns, who gave us so much useful advice for our presentation. We are happy that he is going to be part of our university as Professor of Sustainable Functional Materials starting on December 1st 2021. Also, we got precious assistance from Dr. Nikoline Borgermann who is a biochemist by training and an environmentalist by heart. Further, the Sustainability Office of the TU Darmstadt encouraged us a lot during the preparation of our Coffee Lecture.