On the 18th of March we decided on this years project. We decided to focus on wastewater treatment utilizing a bacterial biofilm. This meeting was not only special because we made this big decision but also because it was our first online meeting during the COVID-19 pandemic. We had to face the fact that we would also have to prepare for online meetings in the future. Furthermore, we had to reckon with a significantly reduced time spend on experiments in the laboratory. To make the best out of this situation we planned to focus on Human Practices, modeling and the theoretical planning of our project. Furthermore, we started the search for sponsors for the upcoming costs.

Since our team mainly consists of first time iGEM participants, we had to start with the question what modeling and Human Practices are and what we could do. Furthermore, we started to plan workshops about general laboratory methods and SnapGene. Additionally, there was a lot more research to conduct. We started looking for a way to enable the biofilm to clean the wastewater.

During our research we came up with the idea of enzymes in the biofilm that should degrade toxic components in wastewater. We thought about a system to first sense these toxic components and then enzymes should be expressed subsequently. This, we observed, would create a super organism able to adapt at any condition. Hence, we argued to not use such system. While looking for problematic micropollutants in wastewater we came up with the idea to engineer EreB to degrade the antibiotic azithromycin.
Our brainstorming about ideas to get in contact with society resulted in the first idea to make a podcast, since this is a safe way to educate others during COVID-19 pandemic. We also started to learn some coding to perform models and thought about what would be interesting and helpful to model in regards to our project idea.

The critical situation due to the COVID-19 pandemic and the fact that we would not have much time in the laboratory caused us to think about how to deal with this and whether we should participate in the iGEM competition at all.

After longer discussions about our participation in iGEM this year, we decided to partake. Considering the shorter laboratory time, we thought about what we could realistically achieve in the laboratory. We at least wanted to clone some enzymes and show that they enable the biofilm to clean wastewater. Furthermore, we brought together some former iGEMers, our advisors and master students from our team to plan the workshops for basic laboratory methods.

We continued to figure out how much laboratory time we would have and what aspects we could manage in the laboratory. While doing this, we also continued to learn some coding for our modeling. Additionally, we stuck to the podcast idea and started to plan how this could be done. To help the team to grow closer together our project monitoring came up with the idea to create online time slots with socializing time for all team members.

This week we started contacting a couple of experts, to get closer into the topic of our project and find out which micropollutants are the most problematic. For modeling we started to plan what and how to model and decided to use RosettaCommons.
During the same week, we had our first socializing meeting online in groups of 3 to 4 people to get to know each other better. We received the important information from the iGEM Headquarter that it is possible to take part in iGEM even if we cannot get into the laboratory. This took away lot of doubts and we focused more on the theoretical planning of our project. But in case we could get into the laboratory, we sought to clone as much enzymes as possible for a proof of concept in E. coli.

Our first modeling approach was to create a ligand that docks to laccase and diclofenac, because according to our research and our first expert talk with Udo Bäuerle this seems to be the most important micropollutant. We therefore worked on a script for this and also on a python script for sequence alignment. The first expert discussion also raised the question on which surface our biofilm should grow. Moreover, we planned to look at the ethical aspects of our project and searched for experts in this field. During our research about more micropollutants to degrade we found out that the laccase can degrade most of them. Luckily, we found some other teams through social media with a similar project.
During all this work our workshops about basic laboratory methods continued and we did online socializing every week.

Our first talk with the iGEM Team Aachen took place and first we talked about our projects and the progress we had made until then. The podcast planning also progressed: we made our own intro melody, thought about a name and a program for the recording. In addition, we prepared for a new expert talk in the next week.
Since we found that the most important micropollutants could be rendered less toxic by the laccase, we focused on this enzyme and started to research how the laccases could be applied in the biofilm most useful. Furthermore, we continued with the idea of enzyme engineering to degrade azithromycin and we searched whether pesticides are a problem too and how to degrade them.

This week we learned that spending time in the laboratory would only be possible with many restrictions, if at all. This meant for us to plan our experiments very well to not waste time, if we could go into the laboratory. To engineer EreB to degrade azithromycin, we talked with Prof. Dr. Viktor Stein about a mutagenesis approach. Moreover, we came up with a possible collabo with Aachen where we wanted to send a paper plane around the world to connect all teams. Additionally, another collaboration about modeling with Hannovercollaboration about modeling with Hannover seemed to be possible.

For the ethical view on our project we sent previously prepared questions to Prof. Dr. Sibylle Gaisser. The same week the iGEM opening weekend festival took place, in which we participated. This really helped to get to know how to deal with the new iGEM rules during the COVID-19 pandemic and we got in contact with many of interesting people.

This week we optimized the docking for the laccase modeling and named our collaboration with Aachen „iJET“. We finally got a name for our podcast and recorded the first episode. For the cloning and testing of the laccase we were looking to design a plasmid and researched for the most suitable laccase for our project. Moreover, we thought about an assay to test the enzyme function. In order to use the laccase in our biofilm, we came up with the idea to immobilize it in the matrix via fusion to TasA (an extracellular matrix protein of the biofilm). The expert Prof. Dr. Susanne Lackner from TU Darmstadt told us about floating bodies on which the biofilm could grow on and then simply be applied in a clarifier.
The iGEM Team from Marburg hosted an online German meetup, in which we wanted to participate. Therefore, we started preparing a poster and two videos to introduce our team and our project to the others.

Our project now took on more shape, as we decided on two suitable laccases, one from Escherichia coli and one from Bacillus subtilis. In order to test how toxic diclofenac and the oxidation product hydroxydiclofenac are, we researched toxicity assays. To engineer EreB for degrading azithromycin, we aimed for a site saturation mutagenesis approach, and to test the activity of the new enzymes, we wanted to perform a Kirby-Bauer Assay. To induce biofilm formation, we thought about the knockout of sinR and to test the stability of the biofilm, we heard about Atomic force microscopy (AFM). So, we started to research for an AFM expert.
To perform the directed mutagenesis in silico, we first wanted to model the structure of EreB. Therefore, as a collaboration with Hannover we thought about a biofilm modeling to further understand the formation and other growth parameters.
Our team decided on a project title and a logo was designed. Additionally, we continued our online socializing and the preparation for the German meetup.

The iGEM Team Duesseldorf hosted as every year a postcard collabo where we wanted do participate. So, we started designing a postcard. The design of our parts and a toxicity assay go on too. The research about pesticides resulted in the recognition, that the German wastewater do not has a problem with pesticides, what were very good news.

Because we wanted to immobilize our enzymes on the biofilm matrix via fusion to TasA, we thought about the knockout of this gene. Therefore, we thought about a proof of concept for the immobilization and came up with the idea, to first test this with GFP, to see, if the protein is still intact while sticking to the biofilm matrix.

Our modeling team started the work on the protein structure of EreB. First, we calculated the primary structure and then continued with the secondary and tertiary structure.

Our Postcard for the Duesseldorf collabo was finished and we ordered them. For the biofilm we considered it would be good to knockout tasA and sinR for the fusion protein und the biofilm formation. But we didn´t know how to do that, so we search for an expert for B. subtilis. To include stakeholders in the development of our project, we talked to Thomas Seeger from the wastewater treatment plant in Weiterstadt.

We talked to Dr. Christian Dietz about the AFM measurement of the biofilm. He also offered us to perform this experiment in his laboratory, which we unfortunately couldn’t do, because of the COVID-19 pandemic. Prof. Dr. Jörg Stülke from Göttingen talked with us about the Bacillus subtilis biofilm and the possible knockouts of tasA and sinR. He also sent us a strain, so we would not have to do the knockout by our own. Because pesticides are no problem we were able to put our time and effort in another topic. Most experts suggested the safe use of our project, which leads to the idea to introduce a kill switch. We also thought about to model this, for getting more information without going into the laboratory.
Looking at Human Practices we got the idea of creating a minigame about general laboratory methods to reach a younger audience. The iJET video tooks shape. We received some videos and together with Aachen started prepare the big video with all of them in it.
We were still looking for some teams to create a partnership. Luckily, we found the iGEM team Stuttgart which project is very close to ours.

The ethical considerations for our project resulted in the aim to make a guidance for the correct use of our project for sewage treatment employees. Considering, that we want to clean the wastewater we decided not to build up a kill switch based on the release of toxins. We would rather use a genetic circuit based on quorum sensing molecules to make the live of the bacterium biofilm dependent.
For our biofilm modeling with Hannover we created a video tool to visualize it. The Minigame got a main character now and we planned some levels. Each level should explain one laboratory method. For our toxicity assay we thought about Vibrio fischeri but we wanted to further research if the best option for this test.
Besides Stuttgart, we got also in contact with Kaiserslautern and started to cooperate together as a bundle of these three teams. Kaiserslautern also invited us to the Wastewater group meeting, where all teams that do something with wastewater treatment came in contact with each other.

We polished our EreB structure with molecular dynamics modeling (MD) and also compared our laccase structures with the laccase structure of Trametes versicolor.

For our kill switch we first got the idea to use two plasmids in two different bacteria to enable them to produce two different essential genes. This makes the live of one bacterium dependent to each other. But this system would require two essential genes and there is the possibility that both types of bacteria leave the wastewater treatment plant together. Additionally, there would be the problem of horizontal gene transfer. So, we decided to do more research about that. While we plan our experiments with the laccases we also researched, what other substances these enzymes could also degrade. One important micropollutant that we were told to be problematic is carbamazepine. So, we continued to collect information about these substances. To simulate the water flow of the wastewater treatment plant, we would like to build a flow chamber and started the research about this.

During all of this we continued to plan new podcast episodes. Additionally, we ran some docking simulations between laccase CotA and the Trametes versicolor laccase for comparison. Because the docking worked better with the T. versicolor laccase we would like to try an enzyme design function of RosettaCommons. The Molecular dynamics modeling with EreB was finished and now we started a docking with azithromycin.

Together with Kaiserslautern and Stuttgart we planned to collect all wastewater treatment information on one page to make it easier or future iGEM Teams to find important information to use for their own projects. Additionally, Kaiserslautern invited us to their Livestream to come and talk with them together about our projects. On the weekend we joined the German meetup, heard amazing talks and get to know other teams. Furthermore, we had the possibility to share our project and to practice an online poster session.

For the toxicity assay we discussed a zebrafish assay but we had to consider very well if an animal experiment is really necessary. Because we animal wouldn’t have the time to perform this assay, we also thought about the need of filling out the iGEM animal use form. For the future development of our project we started designing composite parts consist of TasA and the Enzymes as fusion proteins. Continuing the design of our kill switch we started searching for an essential gene. We came up with the idea to knockout only one essential gene and then reintroduce it while bringing it under the control of a quorum sensing molecule dependent promoter. This makes the live of the bacteria biofilm dependent and solves the previous mentioned problems.

The flow chamber should be printed with a 3D printer. Therefore, we designed the parts to print them. For the videogame we designed the level and wrote explanation texts. For our podcast the second and third episode was scripted. The docking simulations with our enzymes were expanded through other substrates.

To help other iGEM Teams to build their own flow chamber we made DIY modules. About our toxicity assay we decided that a zebrafish assay would be useful, so we planned to fill out the animal use form. Additionally, we discussed the effects of diclofenac on the environment with the ecotoxicologist, Prof. Dr. Jörg Oehlmann. Patrick Schröder from the Federal Environment Agency in Germany, talk with us about the appropriate disposal of medication. Furthermore, me recorded the second and third podcast episode and planned the topics for the next episodes. For the Livestream with Kaiserslautern we prepared a survey, which results we want to discuss live.
For the collaboration with Hannover the first 2D-models of the Biofilm were finished and the modeling for the kill switch started in MATLAB. We continued to model the EreB docking with azythromycin and worked on an easier way to evaluate our modeling results.

This week we started the planning of the wiki and the learning phase of HTML. For our kill switch we thought about dnaA as an essential gene but there must be done more research. Considering the talk with Prof. Dr. Jörg Oehlmann we started thinking about the material of the floating bodies. Because plastic could cause micropollution with microplastic our bisphenol A. For our iJET video the cut was finished and 22 Teams participated. Furthermore, we designed mini-pixel-art for the video game. Because of the upcoming deadline we also selected a track.

To get further improve our project design we hosted a faculty presentation, where staff and professors from the university could give feedback and criticism on our project. Moreover, the Livestream with Kaiserslautern and our risk workshop took place this week.
To integrate the kill switch in the genome of the bacteria we thought about homologous recombination. While doing the kill switch modeling, we realized the lack of date we got to make the model even more realistic. Also, the first kill switch model has been created.

Our first wiki texts were written this week and we thought about, what text we would need for our wiki. In addition, we had an expert talk with PhD Yunrong Chai about microbial genomics and biofilm formation. We got the results from the interview from Kaiserslautern with Dr. Ulrich Ehlers from the German federal office for consumer protection and food safety (BVL), where they also discussed our questions. These results were very important because he explained legal possibilities to establish our project in a real sewage plant. Even if the Livestream was last week, we let the survey open, because we got only 195 participants to that moment. Very exiting was, that we finally uploaded the first podcast episode and the next are on their way. Moreover, we continued to work on our project promotion video.

After long considerations we decided to fill in the safety check-in and the animal use form for the zebrafish embryo toxicity assay. Even if the usage of embryos within five days after spawn does not fall under animal welfare regulations, we found this useful for a responsible planning of our project. For our kill switch modeling we were still searching for experimental data to make the model more specific. The design of our kill switch and the way we want to integrate it into the genome are still not sure, so we planned a talk with Prof. Dr. Torsten Waldminghaus. In addition, we uploaded our second podcast episode. The modeling of the Kill switch iterated into a second model.

This week we thought we could go into the laboratory next week, so we started to prepare everything. Furthermore, we checked WHO and iGEM policy website for further information of safety risk management and made a pro and con list if it is responsible to enable EreB to degrade azithromycin because this would create a new antibiotic resistance. The talk with Prof. Dr. Torsten Waldminghaus was very helpful. Because we got too much homologies in our first plan he suggested to use recombinases. Additionally, he told us that it is possible that the knockout of the essential gene will not kill the cell like a toxin would do. He also stated that dnaA is not the best choice for an essential gene, so we started to research for a new candidate.
For our biofilm model we added adhesion forces between bacteria into our calculations. Therefore, we started looking for real-life values.
Besides the biofilm modelling we refined the second kill switch model into a third one, by adding translation and transcription subunits. Also, a new event function describe the extracellular ComX concentration threshold.

Actually, we would get to the laboratory this week but unfortunately the increased number of new COVID-19 cases made this impossible. Therefore, we focused on the kill switch research about recombinases and a new essential gene. We decided to look mainly at ribosomal proteins. This knockout may not kill the bacteria, but they have problems to separate.
To learn even more about wastewater treatment plants, we visited a wastewater treatment plant and also recorded a little bit for the project promotion video. Finally, our iJET video was finished and published on social media. In view of the advancing time we worked on the wiki texts.

The iGEM year comes closer to the end, what makes us thinking about the last things we have to do to finish our project. Considering our EreB discussion we decided that the engineering of it is reasonable, but to show others that we really thought this through we filled out the check in form for EreB. In order to be able to reach even more listeners we have uploaded our podcast to Spotify.
The modeling collaboration with Hanover goes on. The main work on the code was finished and future work will go into bug fixing.

This week we decided which awards we want to sign in for. The kill switch design goes on and we found an essential gene: the ribosomal protein of the small subunit Rpsb9. The 3D model for our flow chamber is finished and we started the printing of it. Luckily, Prof. Dr. Johannes Kabisch allows us to go into his laboratory with one person to person to test the flow chamber. Our friends from Kaiserslautern planned two more expert interviews, where we could also ask questions, so we prepared this. In addition, we planned an online school visit. Therefore, iGEM Aachen prepared a video about their project which we would share too.

The modelling team build the fusion protein of EreB and TasA in silico. After that a comparative modeling run built the base for a molecular dynamics simulation. This simulations showed us, that our fusion protein would stick together and would not fall apart.
Another part of the modelling team evaluated a modified CotA enzyme design and designed an improved CueO enzyme.

The print of the flow chamber goes on and the fourth podcast episode was recorded. For one of the next episodes we plan to collaborate with iGEM Aachen to share their project too. Our modeling from EreB fusion proteins is simulated and new plots with the biofilm modeling were done. The growth of the bacteria is plotted as a function of iteration steps and the logarithm. With this model we could tell how fast the biofilm grow under defined conditions.

We were still researching for suitable recombinases. The work on our final presentation video started and we did collect ideas how to design it. While writing texts for the wiki the idea to create a safety video came up and we started working on that. Furthermore, we planned the last podcast episodes, which should be two about iGEM, one about CRISPR-Cas, an ethic episode and also one with Aachen.

We had a talk with Prof. Dr. Alfred Nordmann and discussed our project through ethical aspects. We would like to meet with him again and use input for our podcast ethic episode. The flow chamber is ready to test and one person could go into the laboratory next week. Therefore, we prepare some media this week. Mostly we were busy writing wiki texts and planning the final presentation video.

Besides the ongoing work on our wiki we finished the structure for the final presentation video and started designing slides, figures and animations. We managed to be able to go to the laboratory besides the COVID-19 pandemic and are trying to grow a biofilm until next week. We also planned to record a video and pictures of the flow chamber in the following week to show others how to assemble and build their own flow chamber. The last podcast episodes were recorded and we need to redo some figures for the minigame. For our modeling we were in the final steps for the biofilm simulation software.

We had a second interview with Prof. Dr. Alfred Nordmann about ethical aspects. Our friends from Kaiserslautern sent us an e-mail with some answers from Dietmar Schlosser to our questions. Furthermore we talked with Prof. Dr. Ralf Möller and he told us about a possible usage of our biofilm in space travel. Sadly we had some problems with growing our biofilm, so we had to start a new attempt.

We started to work on the final poster for the jamboree and are still working on the wiki. Our online call with a school class took place and we talked about iGEM, SynBio and our research. We also received the last results of our biofilm modeling, which can be found here. Furthermore we finished the last podcasts episodes. Additionally we did the first big rehearsals for the final presentation video and started the recording. Also, we made a slide for the collaboration with iGEM UPCH Peru, to show how our work contributes to the sustainable development goals of the United Nations.