Human Practices for us means to show our project to the public, our excitement for it and to encourage as many people as possible to think about and participate in the discussion regarding synthetic biology and the various aspects linked to it. At the same time, it also means to get external input on how to further improve and adjust our project, solve problems and work together as a team effectively and having fun on the way. On the following page you will see what we have accomplished throughout the year. We hope you will enjoy reading!

This year we chose a project which contains a highly potent neurotoxin: the botulinum toxin - shortly botox. It was inspired by the paper “Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intraneuronal drug delivery” by Prof. Dr. Ichtchenko who showed that three amino acid mutations reduce the toxicity of botulinum toxin C so far to be considered as safe and to be taken from the list of bioweapons. Because of the detoxification we thought it should be possible to work in S1 laboratories and thereby fulfill the iGEM lab safety guidelines. We had to overcome a few obstacles before we were able to do so. For this purpose we stayed in contact with experts of botulinum toxin throughout the year and accompanying to our project. We gained a lot of important knowledge and invaluable advice from the correspondence in this research field. It also offered us the opportunity to modify certain parts of lab work and the chance to restructure our project. Due to the advice of experts like Dr. Binz we learned a lot about how to handle toxic substances in the lab and came up with a safety concept. Through interviews with politicians, medical doctors and scientific researchers who gave us constructive feedback and some new insights we got to reevaluate the potential of our project, possible areas of use and smart additions to it.

Identifying Risk Factors

After we found our project we knew there would be difficulties concerning safety and handling since botulinum toxin is one of the world’s most toxic substances, listed as a potential bioweapon. As we tried to discover how we could work with it nonetheless we came across the possibility to insert specific mutations which render the protein atoxic. Because we new, that there is a working group in the in Institute of Pharmacology and Toxicology of University Hospital Ulm we asked there for expertise support about the handling of AB-Toxins. They referred us to Dr. Thomas Binz a specialist in botox research in Germany, who is currently working at the Hannover Medical School. Luckily he was willing to meet us for an interview and talk about our project idea.

Consequent upon our exchange with Dr. Binz we had a useful talk with Dr. Kittel who is the biosafety officer of our university. We discussed whether we were indeed allowed to work on our project in an S1 environment. It turned out that working with DNA only is appropriate for S1 and that since we’re working only in e.coli we can even transform and express our protein under S1 conditions.

Following the allowance to work in S1 in our university, we talked with our PIs and presented our ideas and how we planned to handle our constructs. At first we ordered the detoxified sequence from IDT which turned out to be very difficult. The GC content was below 30% and even after extreme codon optimization it could not be increased. IDT had big problems synthesizing the genBlocks. After many weeks and just as many failures we finally got a product that unfortunately didn’t fulfill the normal quality standards. In agreement with our PIs it was decided not to use these sequences because they were too precarious due to the whole process of codon optimization as well as the failed synthesis. In addition we could not separate the light chain (LC) and the heavy chain (HC) cleanly from each other. The toxicity of BoNt is provided by the combination of both the HC and LC together. Luckily, we were able to obtain the wt plasmid from Dr. Binz.

Our professors also contacted the regional council. On DNA level, we cleaved the light chain (LC) from the heavy chain (HC) and our PIs stored the HC like the wild type plasmid. We only had access to the in itself atoxic LC. Throughout our project we always handled LC and HC separately. Furthermore the wt plasmid was stored by our professors. After our cleavage work our PIs transformed the plasmid into E.coli in a S2 laboratory to prepare the sample for sequencing. After it was ensured that the cleavage was successful we were able to go on with the rest of our project.

At this point, we were allowed to work with our botox LC with the admission from our PIs, our university and the regional council which is the governmental authority for safety in universities and research in general.

The iGEM Safety Committee

As it is especially important to iGEM that the projects of their contestants are safe we asked the committee whether we were allowed to continue with our project and gave them all our available information. They agreed that once we were able to prove the successful detoxification we could continue to work on it. The only limitation is that our submitted parts with botox will not be included in next years distribution kit due to possible toxicity. We also had to fulfill the iGEMs safety forms thoroughly. Additionally we talked to the iGEM safety officer in person at the European Meetup in Munich. Through this procedure we wanted to completely ensure that we could work with our project safely.

University and Institutes

Our next issue was that we were working in two different labs in two different institutes. We needed a security clearance and talked with Mrs. Dr. Fuss who is responsible for the safety in the cell culture department because our assays needed to be executed with our cell culture and we therefore had to work with botox in the lab. According to her, we were only allowed to work with proteins in her lab but not with DNA. As we ensured the director of the interfaculty institute for biochemistry Prof. Dr. Jansen of the safety of our project an additional concern from another professor came up: we had to be careful of how our project would be represented in the media concerning possible risks.

After assuring our institute director that we thought about all concerned security measures we had to inform him and all other working groups about the major processes of our project including the start of every next step. Next up was contacting Prof. Dr. Stehle who helped in purificating our proteins. As the protein is potentially toxic this was one of the most important aspects. We held a presentation in front of Prof. Dr. Stehle and Dr. Georg Zocher who coached our work and Dr. Christoph Schall who is the biosafety officer of their working group. Together we concluded that the knockout criteria had to be that we should be able to proof by sequencing our protein construct that it had the intended mutations and DNA sequence. Only after validation through our sequencing results we were allowed to transfer our construct in E.coli and express and purify our protein.

The Teammembers

To carry out our project successfully we had an introductory seminar about protein purification held by Dr. Frank Essman. According to him we always have to keep in mind that we are working with a highly toxic protein hence the necessity to apply the highest safety measures in our laboratory work. Of course every lab-team member got a general lab-safety seminar. Additionally we had some group discussion to make sure that everyone works as careful with our botox as if it was still toxic.

To sum up the three most important points you have to keep in mind when working with toxic substances are the following: - First, stay in contact with iGEM so you can consider their advice and guidelines. - Second, clarify everything with your university so that your work takes place under the right circumstances. - And last but not least make sure that all members of the team work with the right mindset and carry out their tasks carefully - with the potentially high risk in the back of their heads.

We started this years Human Practices work with the goal in mind to significantly increase our outreach to the public. By being as present on social media as never before in our iGEM history and documenting and sharing our development and progress on Facebook, Instagram and Twitter along the way we were able to reach iGEM Teams all over the world informing them about our project, iGEM and synthetic biology in general.

The local “Ract!” festival with visitor numbers close to 30,000 was a great first opportunity to implement our goal which we realised by having an informational stand and an additional parcour that was highly frequented by people of all ages. Our public debate in collaboration with our universities debating club Streitkultur e.V. about the topic of gene editing on embryos also turned out to be a huge success due to a very lively and interactive audience that received new insights and perspectives on this highly controversial topic. Because of previous Human Practises projects with schools and their pupils it was dear to our hearts to keep up this tradition, and so we organised a special laboratory day for this next generation of young scientists that was filled with experiments around the detection of Bt corn in corn products.

Last but not least it was very important for us this year to put an emphasis on collaborating and meeting up with other iGEM teams on a national and international level. Whether it was through helping and mentoring the iGEM team Würzburg with the establishment of their team, collaborating with the iGEM team Paris on a very successful bioinformatic prediction tool (“BERT”), meeting up with iGEM team Oslo to present our project and exchange thoughts, advice and ideas, or attending big meet ups like the European or German Meet Up to get in contact with other teams, we are proud to have achieved and exceeded this goal.

If you would like to read more about our this years Public Engagement please visit the page linked below - thank you!