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 Romesberg Lab 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. 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. 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.
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.-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, especially with regards to materials and the workshop.
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.
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, including working materials, expertise, and venues.
Dr. Marcel Frese is currently working at the department of organic chemistry III at Bielefeld University. As part of Dr. Norbert Sewalds team, he provided us with necessary resources to synthetize our own non-canonical amino acid, including working materials, expertise, and venues.
Dr. Sandip Jadhav is currently working at the department of organic chemistry III at Bielefeld University. As part of Dr. Norbert Sewalds team, he provided us with necessary resources to synthesize our own non-canonical amino acid, including working materials, expertise, and venues.
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. 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.
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. Florian Richter is currently working at Bayer in Cologne, Germany, and greatly helped us getting started with the ROSETTA software for our modeling project.
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).
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.
Conference on Expanding the Genetic Code
Figure 1: Excerpt of the program for our workshop EGC: Expanding the Genetic Code.
While the CeBiTec has an excellent reputation as a center for genome research and biotechnology, there is no research related specifically to the expansion of the genetic code. Scientists from the CeBiTec and from Bielefeld University were invited to our workshop. Since there is just basic research on this topic in Bielefeld, the staff benefited from our conference, and gave us very positive feedback regarding the workshop. Moreover, we invited interested students to increase the popularity of iGEM and the knowledge about synthetic biology on the campus of Bielefeld University.
Figure 2: Guest speakers and part of the iGEM team “Bielefeld-CeBiTec 2017”: (left to right, back) Prof. Dr. Nediljko Budisa, Iker Valler Aramburu, Yannic Kerkhoff, Saskia Dymek, Christopher Whitford, (left to right, front) Markus Haak, Michelle Liebers, Olga Schmidt, Denise Kerkhoff
Research in the Budisa Lab, Berlin
Figure 3: From left to right: Huan Sun, Professor Nediljko Budisa, Olga Schmidt and Fabian Schildhauer.
Based on the first positive selection round, problems that may occur in handling the selection process as a whole were explained. The problems compromise for example low transformation rates or low incorporation efficiency of the non-canonical amino acid provided. These particular problems are tried to be eradicated by preparing a fresh batch of competent cells for each selection step and by lowering the concentration of the antibiotic which corresponds to the resistance gene with the blank codon while prolonging the incubation time, respectively.
We decided to study the selection process using two non-canonical amino acids we also use in our toolbox, namely 2-Nitro-L-phenylalanine (2-NPA) and Nε-L-cysteinyl-L-lysin (CL). We were lucky to use existing tyrosyl and pyrrolysyl synthetase libraries provided by the lab, respectively. We were made aware that the selection may not be as efficient as it could be utilizing those libraries, because the synthetases were evolved to bind the distinct non-canonical amino acids we are working with.
Figure 4: Olga at work in Berlin.
Figure 5: Plates with colonies for first positive selection step for 2-NPA. Because 2-NPA is photo cleavable, the plates could not be scanned as usual. (A) and (B) show the plates with added non-canonical amino acid. (C) shows the negative control plate without 2-NPA. Individual colonies could not be counted due to dense growth of the cells.
Figure 6: Plates with colonies for first positive selection step for CL. Plates were scanned. (A) and (B) show the plates with added non-canonical amino acid. (C) shows the negative control plate without CL. Individual colonies could not be counted due to dense growth of the cells.
Figure 7: Plate for first negative selection for CL. Plate was scanned. Colonies are difficult to see due to the plate being scanned.
Figure 8: iGEM Bielefeld-CeBiTec 2017 team members Olga Schmidt and Markus Haak presenting our project in front of the Merck scientists.
Figure 9: iGEM Bielefeld 2017 team members Olga Schmidt and Markus Haak with representatives from the iGEM Bielefeld 2016 team and Merck.
Discussion with Cell Product Purification Experts
Meeting with Prof. Dr. Dirk Lütkemeyer, General Manager of BIBITEC GmbH
Figure 10: 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.
Meeting with Dr. Benjamin Müller, CEO of Biofidus AG
Figure 11: 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.
Meeting with Prof. Dr. Thomas Noll and Ole Weigelt
Figure 12: Meeting with Prof. Thomas Noll and Ole Weigelt Group picture after a beneficial 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.
Meeting with Dr. Florian Richter
Talk on single molecule real time sequencing
Figure 13: Members of the team talking to the invited experts prior to the talk. Interested members of the CeBiTec and our team within the audience.
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”. 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. 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.