Difference between revisions of "Team:Bielefeld-CeBiTec/HP/Gold Integrated"

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         <article>
 
         <article>
 
Due to the innovative nature of our project to expand the genetic code by adding new bases, our team faced novel challenges.  
 
Due to the innovative nature of our project to expand the genetic code by adding new bases, our team faced novel challenges.  
−
One major issue was that this kind of project is far-off topic in our home university. Thus, we were grateful to cooperate with more than twenty scientists from all over the word. This was a great benefit for our work. Besides gaining important insights into important best  
+
One major issue was that this kind of project is far-off topic in our home university. Thus, we were grateful to cooperate with more  
 +
than twenty scientists from all over the word. This was a great benefit for our 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  
 
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. </br>
 
the scientists and partners improved our skills and influenced our work and the planning of our experiments in a great way. </br>
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<div class="section"></div>
 
<div class="section"></div>
 
<p>
 
<p>
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Dr. Sandip Jadhav is currently working at the department of organic chemistry III at Bielefeld University.
+
Dr. Sandip Jadhav is currently working at the department of organic chemistry III at Bielefeld University. As part of Dr. Norbert Sewalds team,
−
As part of a team he provided us with necessary resources to synthesize our own non-canonical amino acid.
+
he provided us with necessary resources to synthesize our own non-canonical amino acid, including working materials, expertise, and venues.
 
</p>
 
</p>
 
<span class="anchor-jump" id="Frese"></span>
 
<span class="anchor-jump" id="Frese"></span>
 
<div class="section"></div>
 
<div class="section"></div>
 
<p>
 
<p>
−
Dr. Marcel Frese is currently working at the department of organic chemistry III at Bielefeld University.
+
Dr. Marcel Frese is currently working at the department of organic chemistry III at Bielefeld University. As part of Dr. Norbert Sewalds team,
−
As part of a team he provided us with necessary resources to synthetize our own non-canonical amino acid.
+
he provided us with necessary resources to synthetize our own non-canonical amino acid, including working materials, expertise, and venues.  
 
</p>
 
</p>
 
<span class="anchor-jump" id="Sewald"></span>
 
<span class="anchor-jump" id="Sewald"></span>
 
<div class="section"></div>
 
<div class="section"></div>
 
<p>
 
<p>
−
Dr. Norbert Sewald is Professor at the faculty of chemistry and head of the department of organic chemistry III at Bielefeld University.
+
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.
+
He and his team provided us with necessary resources to synthetize our own non-canonical amino acid, including working materials, expertise, and venues.
 
</p>
 
</p>
 
<span class="anchor-jump" id="Seidel"></span>
 
<span class="anchor-jump" id="Seidel"></span>
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<div class="section"></div>
 
<p>
 
<p>
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Dr.-Ing. Risto Kõiva is the administrative head of the Bielefeld Excellence Cluster "Cognitive Interaction Technology" (CITEC) workshops.
+
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.
+
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.
 
</p>
 
</p>
  
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  Dr. Budisa</a> from TU Berlin even allowed us to work in his lab to optimize the selection of the amino acyl synthetase.  
 
  Dr. Budisa</a> from TU Berlin even allowed us to work in his lab to optimize the selection of the amino acyl synthetase.  
 
  We incorporated all 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 <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Gold_Integrated#nagoya">Nagoya protocol</a>.
 
  We incorporated all 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 <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Gold_Integrated#nagoya">Nagoya protocol</a>.
−
Beside our main work on expanding the genetic code, working with the general public has been an important part of our project.
+
Beside our main focus on expanding the genetic code, working with the general public has been an important part of our project.
 
  In practice, we organized and held <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Engagement">various events</a>. At the 6th  
 
  In practice, we organized and held <a target="_blank"href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Engagement">various events</a>. At the 6th  
 
  CeBiTec students’ academy and the Teutolab, we taught the basic methods of synthetic biology and bioinformatics to people of all ages.  
 
  CeBiTec students’ academy and the Teutolab, we taught the basic methods of synthetic biology and bioinformatics to people of all ages.  
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  experiment with every-day materials.  
 
  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.</br>
 
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.</br>
−
The following sections highlight our interactions with experts and how they shaped our project.  
+
The following sections highlight our interactions with experts and how they influenced our project. They not only shaped our project,
 +
but also broadened our horizon on how to communicate and interact with the public about our future research in our coming careers.  
  
 
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</div class="article">
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<article>
 
<article>
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While synthetic biology offers solutions to many current issues, it has also been met with skepticism with regards to its responsible  
+
While synthetic biology offers solutions to many current issues, it has also been met with scepticism with regards to its responsible  
 
use and implications resulting from its research. To assess chances and implications of an expanded genetic code in particular, we interviewed  
 
use and implications resulting from its research. To assess chances and implications of an expanded genetic code in particular, we interviewed  
 
experts from the scientific, philosophical, and religious fields. Furthermore, the public was asked for their opinions regarding this topics in a survey.
 
experts from the scientific, philosophical, and religious fields. Furthermore, the public was asked for their opinions regarding this topics in a survey.
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  <a target="_blank"href="https://en.wikipedia.org/wiki/Nediljko_Budisa">Nediljiko “Ned” Budisa</a> is a biochemist and full professor of bio catalysis at the Technical University of Berlin, Germany. He is not just an expert regarding chemical synthetical biology (xenobiology), but also on genetic code engineering, making him an excellent adviser for our project. Ned talked about “the potentials of non-canonical amino acids in synthetic biology  and possible alternative life”. His talk gave a broad overview about the field. He put emphasis on the fact that the genetic code is degenerated and for incorporating non-canonical amino acids, an extension of the genetic code is needed. This extension could be achieved by evolution and selection of tRNA/amino acyl-tRNA synthetase pairs. This pair needs to be chosen depending on the chemical properties and conformation of the given non-canonical amino acids to ensure maximal substrate enzyme binding and incorporation efficiency.   
 
  <a target="_blank"href="https://en.wikipedia.org/wiki/Nediljko_Budisa">Nediljiko “Ned” Budisa</a> is a biochemist and full professor of bio catalysis at the Technical University of Berlin, Germany. He is not just an expert regarding chemical synthetical biology (xenobiology), but also on genetic code engineering, making him an excellent adviser for our project. Ned talked about “the potentials of non-canonical amino acids in synthetic biology  and possible alternative life”. His talk gave a broad overview about the field. He put emphasis on the fact that the genetic code is degenerated and for incorporating non-canonical amino acids, an extension of the genetic code is needed. This extension could be achieved by evolution and selection of tRNA/amino acyl-tRNA synthetase pairs. This pair needs to be chosen depending on the chemical properties and conformation of the given non-canonical amino acids to ensure maximal substrate enzyme binding and incorporation efficiency.   
 
</br>
 
</br>
−
Through the following discussions regarding our project, Ned’s insight into our project was very important. Given that we want to incorporate non-canonical amino acids into proteins and peptides, he made us aware that we need to look into the formation and capacity of the binding pocket of the synthetases we want to evolve and select for the non-canonical amino acids. He also indicated possible problems for in vivo incorporation of non-canonical amino acids, mainly the elongation factor EF-Tu and the ribosomes. On the topic of unnatural base pairs and incorporation of non-canonical amino acids, Ned gave us the advice to consider using the synthetase for pyrrolysin. This specific synthetase does not recognize the anticodon loop on its corresponding tRNA. Thus, the changing of the codon and anticodon would possibly not interfere with ribosomal incorporation of a given amino acid using unnatural base pairs. </br>
+
Through the following discussions regarding our project, Ned’s insight into our project was very important. Given that we want to incorporate non-canonical amino acids into proteins and peptides, he made us aware that we need to look into the formation and capacity of the binding pocket of the synthetases we want to evolve and select for the non-canonical amino acids. He also indicated possible problems for <i>in vivo</i> incorporation of non-canonical amino acids, mainly the elongation factor EF-Tu and the ribosomes. On the topic of unnatural base pairs and incorporation of non-canonical amino acids, Ned gave us the advice to consider using the synthetase for pyrrolysin. This specific synthetase does not recognize the anticodon loop on its corresponding tRNA. Thus, the changing of the codon and anticodon would possibly not interfere with ribosomal incorporation of a given amino acid using unnatural base pairs. </br>
 
We are very happy that Ned invited us to his laboratory in Berlin. Our team member Olga went to Berlin to work with experts on this research field and to use a sophisticated selection library for the generation of a novel tRNA synthetase. </br>
 
We are very happy that Ned invited us to his laboratory in Berlin. Our team member Olga went to Berlin to work with experts on this research field and to use a sophisticated selection library for the generation of a novel tRNA synthetase. </br>
 
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On the first day, the theoretical background of evolution and selection of the amino acyl-tRNA synthetases was discussed. This step was necessary because many Labs evolve the synthetases according to the non-canonical amino acids they want to utilize, and various selection plasmids and protocols are used for selection processes. <br>
+
On the first day, the theoretical background of evolution and selection of the amino acyl-tRNA synthetases was discussed. This step was necessary because many labs evolve the synthetases according to the non-canonical amino acids they want to utilize, and various selection plasmids and protocols are used for selection processes. <br>
 
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 resistence gene with the blank codon while prolonging the incubation time, respectively. <br>
 
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 resistence gene with the blank codon while prolonging the incubation time, respectively. <br>
 
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. <br>
 
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. <br>
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                <div class="figure large">
 
                <div class="figure large">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/e/e1/T--Bielefeld-CeBiTec--2NPA-POS.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/e/e1/T--Bielefeld-CeBiTec--2NPA-POS.png">
−
                         <p class="figure subtitle"><b>Figure 5:Plates with colonies for first positive selection step for 2-NPA. </b> 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.<p>
+
                         <p class="figure subtitle"><b>Figure 5: Plates with colonies for first positive selection step for 2-NPA. </b> 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.<p>
 
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</div>
 
</div>
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<div class="figure large">
 
<div class="figure large">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/a/ae/T--Bielefeld-CeBiTec--CL-POS.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/a/ae/T--Bielefeld-CeBiTec--CL-POS.png">
−
                         <p class="figure subtitle"><b>Figure 6:Plates with colonies for first positive selection step for CL. </b> 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.<p>
+
                         <p class="figure subtitle"><b>Figure 6: Plates with colonies for first positive selection step for CL. </b> 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.<p>
 
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</div>
 
</div>
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                 <article>
 
                 <article>
−
One negative selection round was also performed for CL by Huan Sun (Figure 7). Thought not all of the needed three positive and two negative selection rounds could be performed, the preliminary results seem to be promising. To make sure if the non-canonical amino acid was incorporated through amber codon suppression, Mass Spectrometry needs to be performed. <br>
+
One negative selection round was also performed for CL by Huan Sun (Figure 7). Though not all of the needed three positive and two negative selection rounds could be performed, the preliminary results seem to be promising. To make sure that the non-canonical amino acid was incorporated through amber codon suppression, Mass Spectrometry needs to be performed. <br>
 
</article>
 
</article>
 
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                 <article>
 
                 <article>
−
We thank the whole Budisa Lab, Professor Budisa, Dr. Tobias Baumann, Huan Sun and Fabian Schildhauer for their help, interesting discussions and useful advice even after Olgas stay in Berlin. <br>
+
We thank the whole Budisa Lab, Professor Budisa, Dr. Tobias Baumann, Huan Sun and Fabian Schildhauer for their help, interesting discussions and useful advice also after Olgas stay in Berlin. <br>
  
 
         </article>
 
         </article>
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<h2 id="EluXExperts"> Discussion with Cell Product Purification Experts </h2>
 
<h2 id="EluXExperts"> Discussion with Cell Product Purification Experts </h2>
 
                 <article>
 
                 <article>
−
                     Feedback and advice from experts actually working in the fields of concern are valuable contributions to a successful iGEM project. To get advice on how to improve our <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">purification column EluX</a>, 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.                 
+
                     Feedback and advice from experts actually working in the fields of concern are valuable contributions to a successful iGEM project. To get advice on how to improve our <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">purification column EluX</a>, 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.                 
 
                 </article>
 
                 </article>
 
<h3> Meeting with Prof. Dr. Dirk Lütkemeyer, General Manager of BIBITEC GmbH  </h3>
 
<h3> Meeting with Prof. Dr. Dirk Lütkemeyer, General Manager of BIBITEC GmbH  </h3>
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He also pointed out some things we will have to check if a real use of this elution technique becomes current. 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.  
+
He also pointed out some aspects we will have to check if a real use of this elution technique becomes current. 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.  
 
</article>
 
</article>
 
</div>
 
</div>

Revision as of 21:00, 1 November 2017

Integrated Human Practice

Short summary

Due to the innovative nature of our project to expand the genetic code by adding new bases, our team faced novel challenges. One major issue was that this kind of project is far-off topic in our home university. Thus, we were grateful to cooperate with more than twenty scientists from all over the word. This was a great benefit for our 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.
The figure below displays the most influential experts we contacted throughout our work. You can click on each name to receive some information on who they are and how they helped us.
Browser cannot display svg images!

Experts

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 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. 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. 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. 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, especially with regards to materials and the workshop.

Overview

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. Prof. Dr. Budisa from TU Berlin even allowed us to work in his lab to optimize the selection of the amino acyl synthetase. We incorporated all 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 focus 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.
The following sections highlight our interactions with experts and how they influenced our project. They not only shaped our project, but also broadened our horizon on how to communicate and interact with the public about our future research in our coming careers.

ChImp Report

While synthetic biology offers solutions to many current issues, it has also been met with scepticism with regards to its responsible use and implications resulting from its research. To assess chances and implications of an expanded genetic code in particular, we interviewed experts from the scientific, philosophical, and religious fields. Furthermore, the public was asked for their opinions regarding this topics in a survey. On the basis of our research, we developed practical guidelines for research projects concerning synthetic biology, some of which we ourselves applied during our iGEM project. Evaluating all perspectives, we developed flexible and easily applicable multi-faceted guidelines for research concerning synthetic biology, such as the incorporation of expert committees relevant for the project, as well as possible dual use concerns. The complete and comprehensive Report on Chances and Implications of an Expanded Genetic Code (ChImp Report) can be accessed here.

Conference on Expanding the Genetic Code

From the beginning, we knew that our project is multi-facetted, and that we would need the insight and expertise of experienced scientists. Therefore, we organized our own open workshop, “EGC: Expanding the Genetic Code”, on August 4th 2017 to obtain some advice and tips for our work. We did not just intend to broaden our horizon regarding the possibilities and challenges of our project, but also to get feedback and help from external experts in synthetic biology.

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.
After the official part of the workshop, we had an intense exchange with two of our invited speakers. Iker Valle Aramburu (EMBL Heidelberg, Germany) received a degree in Biochemistry at the University Basque Country, Spain and his MSc in Cell Biology at Heidelberg University. His field of study includes Physical Chemistry, Chemical Kinetics, and Biochemistry. His presentation dealt with “the plasticity of FG-Nucleoporins and NTR interactions”. Due to his experience with small molecular FRET, Mr. Aramburu gave us pointers for our analyzing tool. Nediljiko “Ned” Budisa is a biochemist and full professor of bio catalysis at the Technical University of Berlin, Germany. He is not just an expert regarding chemical synthetical biology (xenobiology), but also on genetic code engineering, making him an excellent adviser for our project. Ned talked about “the potentials of non-canonical amino acids in synthetic biology and possible alternative life”. His talk gave a broad overview about the field. He put emphasis on the fact that the genetic code is degenerated and for incorporating non-canonical amino acids, an extension of the genetic code is needed. This extension could be achieved by evolution and selection of tRNA/amino acyl-tRNA synthetase pairs. This pair needs to be chosen depending on the chemical properties and conformation of the given non-canonical amino acids to ensure maximal substrate enzyme binding and incorporation efficiency.
Through the following discussions regarding our project, Ned’s insight into our project was very important. Given that we want to incorporate non-canonical amino acids into proteins and peptides, he made us aware that we need to look into the formation and capacity of the binding pocket of the synthetases we want to evolve and select for the non-canonical amino acids. He also indicated possible problems for in vivo incorporation of non-canonical amino acids, mainly the elongation factor EF-Tu and the ribosomes. On the topic of unnatural base pairs and incorporation of non-canonical amino acids, Ned gave us the advice to consider using the synthetase for pyrrolysin. This specific synthetase does not recognize the anticodon loop on its corresponding tRNA. Thus, the changing of the codon and anticodon would possibly not interfere with ribosomal incorporation of a given amino acid using unnatural base pairs.
We are very happy that Ned invited us to his laboratory in Berlin. Our team member Olga went to Berlin to work with experts on this research field and to use a sophisticated selection library for the generation of a novel tRNA synthetase.

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

At the end of September, our team member Olga went to Berlin to work within the Budisa Lab to learn more about distinct methodological approaches needed for the project, mainly to practice the handling of the selection of the aminoacyl-tRNA synthetases, at the invitation of Professor Nediljiko “Ned” Budisa during our Workshop “Expanding the Genetic Code”.

Figure 3: From left to right: Huan Sun, Professor Nediljko Budisa, Olga Schmidt and Fabian Schildhauer.

On the first day, the theoretical background of evolution and selection of the amino acyl-tRNA synthetases was discussed. This step was necessary because many labs evolve the synthetases according to the non-canonical amino acids they want to utilize, and various selection plasmids and protocols are used for selection processes.
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 resistence 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.

During the handling of each first positive selection step, Olga was supervised by Huan Sun (pylRS) and Fabian Schildhauer (tyrRS).

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.

One negative selection round was also performed for CL by Huan Sun (Figure 7). Though not all of the needed three positive and two negative selection rounds could be performed, the preliminary results seem to be promising. To make sure that the non-canonical amino acid was incorporated through amber codon suppression, Mass Spectrometry needs to be performed.

Figure 7: Plate for first negative selection for CL. Plate was scanned. Colonies are difficult to see due to the plate being scanned.

We thank the whole Budisa Lab, Professor Budisa, Dr. Tobias Baumann, Huan Sun and Fabian Schildhauer for their help, interesting discussions and useful advice also after Olgas stay in Berlin.

Merck

Figure 8: iGEM Bielefeld-CeBiTec 2017 team members Olga Schmidt and Markus Haak presenting our project in front of the Merck scientists.

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 continuous 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.
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 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

Feedback and advice from experts actually working in the fields of concern are valuable contributions to a successful iGEM project. To get advice 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 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.

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 focusing 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.
He also pointed out some aspects we will have to check if a real use of this elution technique becomes current. 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.

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

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 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.

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, shareing 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 degree 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.

Talk on single molecule real time sequencing

One of the problems we had to face when dealing with unnatural bases was how to detect them in sequence analysis. However, there were many different sequencing methods that we had to consider. One of these is the single molecule real time sequencing that for instance is offered by pacific biosciences. For this purpose, we invited representatives of the company to introduce the method as well as data analysis patterns to us. The 5th of September, 2017, at 5pm, Mr. Cong Luan Nguyen as well as Mr. David Stucki gave a talk on “Single Molecule Real Time Sequencing with the Sequel System and an outlook in data analysis”. This talk was attended by our team as well as many interested colleagues. It was concluded with interesting discussion featuring different aspects of the technology. The talk highlighted why this system is good to be used with unnatural base pairs but also showed advantages of a general usage at the CeBiTec. Therefore, all in all, we gained some more insights into sequencing with the new technology and possible usages for our project.

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.

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.