Team:Bielefeld-CeBiTec/HP/Gold Integrated

Integrated Human Practice

Due to the innovative nature of our project to expand the genetic code by adding new bases, our team faced very novel challenges. Due to some lack of expertise regarding specific details of our project at our university, the cooperation with more than twenty scientists from all over the world was a great enrichment for our own work. Besides gaining important insights into important best practices, we kept in contact to leading experts to evaluate if our project can improve basic research methods. The feedback from the scientists and partners improved our skills and influenced our work and the planning of our experiments in a great way. Prof. Dr. Budisa from TU Berlin even allowed us to work in his lab to optimize the selection of the amino acyl synthetase. Our project involves a great amount of multidisciplinarity and touches topics of chemistry (synthesis of our own designed amino acid), electrical engineering (design and construction of our LED-Panel), synthetic biology, biosafety issues (Biosafety report) and awareness of consequences of our own research (ChImp report). Some of the scientists we contacted participated in our workshop ‘Expanding of the Genetic Code’. There, we received a lot of feedback for our project and obtained valuable information, but also learned about ethical concerns regarding our approach to incorporate new bases. We incorporated these insights by working with the general public and numerous experts in ethics and prepared a report about ‘Chances and Implications of an expanded genetic code’, where we draft guidelines for the handling of unnatural bases. Working with a foreign plant gave us the chance to learn about the laws and guidelines of the Nagoya protocol. Beside our main work on expanding the genetic code, working with the general public has been an important part of our project. In practice, we organized and held various events. At the 6th CeBiTec students’ academy and the Teutolab, we taught the basic methods of synthetic biology and bioinformatics to people of all ages. At the GENIALE, a science event with booths all over the city, we did exciting experiments with visitors and showed them how to conduct experiment with every-day materials. The industrial usage can be an important factor for the design of applications. We had the chance to cooperate with MERCK, Biofidus AG, BIBITEC GmbH and an attorney for patent law, where we presented our project and got important feedback with regards to our approach on the unnatural bases and our applications.

Discussion with Cell Product Purification Experts

Feedback and advice from experts actually working in the fields of concern are valuable contributions to a successful iGEM project. To get advise on how to improve our purification column EluX, we met with two experts: Prof. Dirk Lütkemeyer, General Manager of BIBITEC GmbH, and Dr. Benjamin Müller, CEO of Biofidus AG for Analytical Services. BIBITEC GmbH was founded in 2001 and specializes in the production of recombinant proteins and monoclonal antibodies. Biofidus AG is a bioanalytical company offering services with a wide variety of bioanalytical methods. They are specialized on spectroscopic, chromatographic or mass spectrometric assays focused on characterization of proteins as well as small molecules. We demonstrated and explained our prototypes and elution technique. We also prepared specific questions, such as ‘what do you think about our elution technique compared to common techniques?’ and ‘Which problems and limitations could our column encounter in real world applications?’. As described in the following sections, we benefited greatly from these exchanges and made several improvements to our hardware to address the predicted challenges. Therefore, we are very thankful for their opinions and great advices.

Meeting with Prof. Dr. Dirk Lütkemeyer, General Manager of BIBITEC GmbH

We met Prof. Dirk Lütkemeyer at the 24th of August at the Center for Biotechnology. We presented our purification column and explained our light-induced elution technique. The presentation was followed by a fruitful discussion about possible challenges in real world applications, like using this purification technique to purify recombinant proteins produced by our local fermentation technologies working group.

Figure 1: Meeting with Prof. Lütkemeyer.
Prof. Dirk Lütkemeyer (BIBITEC GmbH) and team member Yannic during a meeting at the Center for Biotechnology. A detailed discussion provided us with useful advices and opinions about our elution technique and purification column.

Prof. Lütkemeyer stated that our technique of the light-induced elution is very interesting and a promising alternative to proteases-cleaving sites. However, we need to validate the system profoundly. Especially interesting are the minimal amounts of light and radiation time needed for reliable back bone cleavage. He also said that our system could be very useful for companies related to the production of biomolecules like Evonik Industries AG and GE Healthcare. Nevertheless, we should improve our purification column prototype design to reduce dead space, increase potential yield and enhance the scalability. Finally, he recommended to focus on the validation of our elution system, since this is a highly innovative and promising aspect.

Meeting with Dr. Benjamin Müller, CEO of Biofidus AG

We met Dr. Benjamin Müller (CEO of Biofidus AG) on the 6th of September at his office (Figure 2). We showed him our peptide purification column and discussed the possibilities and conceivable use cases of our light-induced elution technique. He commented on the advantage of our purification method, namely that the one step reaction is simple and easy to understand and thus attractive for possible users.

Figure 2: Meeting with Dr. Benjamin Müller
Meeting of Yannic Kerkhoff with Dr. Benjamin Müller at his office on the 6th of September to discuss the light-induced elution technique.

He also pointed out some things we will have to check if we want to proclaim a real use of this elution technique. He stated that we need to investigate if the light that is used for the elution will not harm the target protein and if there are any restrictions to the light-spectra, intensity, or irradiation time. We should also validate the stability of the amino acid itself, namely if it only reacts after irradiation, or possibly in the storage process. This would lead to a noticeable loss of the target protein. Furthermore, he stated that it would be useful to create a kit for the light-induced elution so that all users can apply this technique to their desired process and make the usage as easy as possible.

Meeting with Prof. Dr. Thomas Noll and Ole Weigelt

To discuss possible marketing and commercialization strategies for our EluX technology, we were invited to a meeting by Prof. Thomas Noll, co-founder of the local biotech Xell AG, and Ole Weigelt, tax consultant and lawyer at Weigelt Miersbach Uhlemeyer Partnerschaftsgesellschaft. The meeting took place on the 7th of September at Ole Weigelt’s office.

Figure 3: Meeting with Prof. Thomas Noll and Ole Weigelt
Group picture after a fruitful meeting about possible marketing and commercialization strategies for our EluX technology.From left to right: Yannic Kerkhoff, Prof. Thomas Noll (co-founder of Xell AG), Ole Weigelt (co-founder of Xell AG, lawyer and tax consultant) and Christopher Whitford.

Prof. Noll and Mr. Weigelt both stated that our idea has a high innovation potential and could be used as a new purification method. As examples they named the very fragile factor VIII and proteins A and G due to their expensive column material. Since the iGEM idea is based on making your research open source and share your results with the whole community, Prof. Noll and Mr. Weigelt said that an intense scientific analysis of the method is more adequate as commercializing it. Both encouraged us to pursue our idea properly and to continue the work even after the competition either by ourselves or by handing over our results to other students of our university, who could analyze this technique in bachelor or master theses.
Both experts pointed out how multifaceted our concept is, as it includes basic molecular biology, cell development, photometry, linker design, cleavage kinetics and media optimization. Eventually, Mr. Weigelt and Prof. Noll advised us in testing the range of wave lengths and light intensities suitable for the cleavage of the target protein without harming either the target protein or the affinity tag. We thus started some investigations into how the UV-light could potentially harm our proteins.

Nagoya-Protocol

The “Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization (ABS) to the Convention on Biological Diversity” entered into force on October 12th, 2014 in Nagoya. It is an international environmental agreement to implement the objectives of the 1993 UN Convention on biological diversity: “the fair and equitable sharing of benefit arising out of the utilization of genetic resources” [2]. Especially developing countries which often have a manifold biodiversity are affected by bio piracy. The Nagoya Protocol intends to ensure that these countries at least profit financially or non-financially by the resulting research and products of their genetic resources. In addition to accessing genetic resources, it covers traditional knowledge concerning these resources. Contracting countries are bound to take action to guarantee informed consent prior to any course of action, as well as impartial benefit-sharing, respecting local laws as well as customary use and exchange.

Today, 100 countries signed the Nagoya Protocol [3]. To succeed, every partner nation should establish ABS National Focal Points, competent national authorities, legislative administrative, national databases, and checkpoints for information.

Germany signed the Nagoya protocol on July 20th, 2016. Our donor of the isoG-metabolism-pathway is Croton tiglium, which is a herb in traditional Asian medicine. Its original occurrence is in the Asian region. To make sure we do not infringe upon the Nagoya Protocol regulations, we clarified the origin of our plant as we got it from the botanical garden of the Phillips University Marburg. Croton tiglium is originally from an undocumented old stock of the botanical garden Giessen. In 1986, the botanical garden Marburg received seeds from this stock. The date on which the EU regulation entered into force was October 2014. All resources collected before this date are not subject to reporting requirements of the Nagoya Protocol in Germany. Thomas Greiber (Federal Agency for Nature Conservation, Head of Division Div I 1.4 “Competent National Authority for the Nagoya Protocol”) confirmed “[..]Therefore, the respective areas of application are not touched by this EU-regulation, and the mentioned regulations are not relevant.”

Therefore, we are allowed to work with the plant and publish information and results about gene sequences and metabolism pathways without violating applicable law.

Merck

Merck is a leading company for products in the pharmaceutical and chemical sectors. Their expertise consists of three mayor sectors: Healthcare, Life Science and Performance Materials. This wide variety of subject areas ensures Merck's status as a global player. Their close collaboration with scientists ensures the high-quality of their products and know-how. Merck has supported the iGEM Bielefed-CeBiTec teams for seven years. We are grateful for their continues scientific support and expertise. Back in August, we visited Merck to present our final project idea and the first results. Our visit was organized by Prof. Herget, who has been a great supporter of the iGEM-Teams Bielefeld for several years.

Figure 2: iGEM Bielefeld-CeBiTec 2017 team members Olga Schmidt and Markus Haak presenting our project in front of the Merck scientists.

The following discussion dealt particularly with possible in vitro and in vivo approaches and the ethical concerns regarding our project. They confirmed our idea to add in vitro experiments as an additional control for our project. The moral concers have also been addressed. Therefore, we designed a survey and asked for second opinions in a variety of meetings with ethicists. This eventuated in our report “ChImp - Chances and Implication of an Expanded Genetic Code”, from which we derive guidelines and recommendations for work awareness in science. Visiting Merck is valuable in more than just one way: we did not just gain experience in holding a presentation in front of experienced researchers, but also received great advice for the future development of our project. We are excited to visit Merck again in 2018 to present our final results and the progress we have made.

Figure 3: iGEM Bielefeld 2017 team members Olga Schmidt and Markus Haak with representatives from the iGEM Bielefeld 2016 team and Merck.

Experts Overview

Dr. Martin Smith is a computational biologist and head of genomic technologies at the Garvan Institute of Medical Research in Sydney, Australia. His research revolves around biological mechanisms that control how genes are activated and repressed. Dr. Martin Smith is also an expert in Oxford Nanopore Sequencing and gave us very helpful information for sequencing of very low amounts of DNA and single cell sequencing using Oxford Nanopore Sequencing.

Prof. Dr. Thomas Carell is a German chemist and head of the research group Organic and Biomolecular Chemistry at the Ludwig-Maximilians-University Munich. His research focusses on the use of modern synthetic organic chemistry to synthesize naturally occurring modified DNA and RNA bases and to study how chemistry on DNA and RNA bases influences life. Prof. Dr. Thomas Carell is one of the leading experts on DNA and RNA chemistry in Germany.

Prof. Dr. Dirk Lütkemeyer is the General Manager of BIBITEC GmbH. He gave us helpful advice concerning the construction and further development of the purification column eluX.

Dr. Benjamin Müller is the CEO of Biofidus AG, a bioanalytical company located in Bielefeld, Germany. During our meeting we discussed the pros and cons of the light-induced elution method. Furthermore, Dr. Benjamin Müller helped us with several questions about analytics of biomolecules.

Prof. Dr. Thomas Noll is cofounder of Xell AG and professor for cell culture technology at Bielefeld University. Together with Ole Weigelt, he advised us on the possible commercialization of our light-induced elution method and provided some insight into which companies might be interested in such a technology.

Ole Weigelt is tax consultant and lawyer at Weigelt Miersbach Uhlemeyer joint venture partner. Together with Prof. Thomas Noll, he advised us on the possible commercialization of our light-induced elution method and provided some insight into which companies might be interested in such a technology.

Raul Machado is an Assistant Professor at the University of Minho, Portugal, where he focusses on genetically engineered protein-based materials. He gave us advice on finding a method to build silk elastin like proteins by recursive directional ligation (pre RDL).

Dr. Nediljko Budisa is a Professor at the TU Berlin, Germany, at the institute for chemistry. He provided us with valuable information on the aaRS evolution process and gave us access to his lab for one week to work on our project.

Dr. Florian Richter is currently working at Bayer in Cologne, Germany, and greatly helped us getting started with the ROSETTA software for our modeling project.

Iker Valle Aramburu is a predoctoral fellow at the EMBL Heidelberg, Germany. He gave us valuable initial information on labeling non-canonical amino acids.

Dr. Sandip Jadhav is currently working at the department of organic chemistry III at Bielefeld University. As part of a team he provided us with necessary resources to synthetize our own non-canonical amino acid.

Dr. Marcel Frese is currently working at the department of organic chemistry III at Bielefeld University. As part of a team he provided us with necessary resources to synthetize our own non-canonical amino acid.

Dr. Norbert Sewald is Professor at the faculty of chemistry and head of the department of organic chemistry III at Bielefeld University. He and his team provided us with necessary resources to synthetize our own non-canonical amino acid.

Dr. Thorsten Seidel is head of the research group Dynamic Cell Imaging at the faculty of biology at Bielefeld University. He provided us with information on the fluorescence microscope and expertise in bio imaging and the FRET system.

Prof. R. Alta Charo is a professor of law and bioethics at the University of Wisconsin Law School, USA. She gave us valuabel opinions on chances and implications of an expanded genetic code from an ethical and philosophical perspective.

Thomas Greiber is working at the Bundesamt für Naturschutz (Federal Office of Nature Protection) in Bonn, Germany. He gave a talk on the Nagoya Protocol, which was relevant for our work.

During the Labvolution Exhibition in Hannover, Germany, we were able to get in contact with many experts in the field and gather first information relevant for our topic.

Dr. Vitor Pinheiro is a lecturer in synthetic biology and at the ISMB in London, UK, and leader of the Pinheiro Lab. With his expertise, he gave us initial advice regarding our project as a whole.

Dr. Piet Herdewijn is a professor at the faculty of pharmaceutical sciences at the KU Leuven, Belgium. With his expertise, he gave us initial advice regarding our project as a whole.

Dr. Mario F. Feldman is an associate professor of molecular microbiology at the Washington University in St. Louis, USA While deciding on a project topic, he helped us with research with regards to the glycosylation in E. coli.

Dr. Floyd E. Romesberg is professor of chemistry and head of the RomesbergLab at the Scripps Research Institute in California, USA. During the beginning of our project, he gave us motivating advice on our project in general.

Dr. Melanie Schwarz is an account manager at Biolegio. She helped us with regards to oligonucleotides containing unnatural bases and gave us information on annealing and quality control of our work.

Dr.-Ing. Risto Kõiva is the administrative head of the Bielefeld Excellence Cluster "Cognitive Interaction Technology" (CITEC) workshops. He answered our questions with regards to the hardware design and provided resources for building our hardware.