iGEM 2020 — Team TU_Darmstadt

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TU Darmstadt/Notebook

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[![logo](https://static.igem.org/mediawiki/2020/4/47/T--TU_Darmstadt--black_owl.png)](https://2020.igem.org/Team%3ATU_Darmstadt)

* PROJECT

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Description)[BACKGROUND](https://2020.igem.org/Team%3ATU_Darmstadt/Background)
  [IMPLEMENTATION](https://2020.igem.org/Team%3ATU_Darmstadt/Implementation)[PHARMACEUTICAL DEGRADATION](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation)
  [BIOFILM](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm)
  [KILL SWITCH](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Kill_Switch)[NOTEBOOK](https://2020.igem.org/Team%3ATU_Darmstadt/Notebook)
  [EXPERIMENTS](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments)
  [BACKBONES](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Backbones)
* PEOPLE

  [TEAM](https://2020.igem.org/Team%3ATU_Darmstadt/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Attributions)
  [COLLABORATIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations)
  [SPONSORING](https://2020.igem.org/Team%3ATU_Darmstadt/People/Sponsoring)
* MODELING

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Model)[ENZYME MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling)
  [BIOFILM MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Biofilm_Modeling)
  [KILL SWITCH MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling)[ROSETTA GUIDE](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Rosetta_Guide)
* HUMAN PRACTICES

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Human_Practices)[INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices)
  [SCIENCE COMMUNICATION & EDUCATION](https://2020.igem.org/Team%3ATU_Darmstadt/Education)
  [RESPONSIBLE RESEARCH](https://2020.igem.org/Team%3ATU_Darmstadt/HP/Ethics)
* PARTNERSHIP

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)[SUMMARY & METHODS](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Summary_And_Methods)
  [EXPERTS & LITERATURE](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Experts_And_Literature)
* SAFETY

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Safety)[HOW TO HANDLE B-TOX](https://2020.igem.org/Team%3ATU_Darmstadt/Safety/How_To_Handle_B-Tox)
* ACHIEVEMENTS

  [AWARDS](https://2020.igem.org/Team%3ATU_Darmstadt/Awards)[BEST SOFTWARE](https://2020.igem.org/Team%3ATU_Darmstadt/Software)
  [BEST SUSTAINABLE DEVELOPMENT IMPACT](https://2020.igem.org/Team%3ATU_Darmstadt/Sustainable)[PARTS](https://2020.igem.org/Team%3ATU_Darmstadt/Parts)
  [CONTRIBUTION](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ATU_Darmstadt/Engineering)
* JUDGING FORM

  [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3429)[POSTER](https://2020.igem.org/Team%3ATU_Darmstadt/Poster)

# Notebook

[March](#March)
[April](#April)
[May](#May)
[June](#June)
[July](#July)
[August](#August)
[September](#September)
[October](#October)

[March](#March)
[April](#April)
[May](#May)
[June](#June)
[July](#July)
[August](#August)
[September](#September)
[October](#October)

## Our Journey

Here you can follow the **progress of our project for the last 32 weeks**. This description of our journey helps you **understand the development of our project**.

Unfortunately, the COVID-19 pandemic lead to many troubles but we managed to overcome most of them and **plan our project theoretically**. We focused on  [modeling](https://2020.igem.org/Team%3ATU_Darmstadt/Model), [Human Practices](https://2020.igem.org/Team%3ATU_Darmstadt/Human_Practices) and the research aspects of our project. To keep safe, we also developed **new ideas to grow together as a team online**.

TOGGLE ALL

## March

Week 12

On the 18th of March **we decided on this years  [project](https://2020.igem.org/Team%3ATU_Darmstadt/Description)**. 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](https://2020.igem.org/Team%3ATU_Darmstadt/Human_Practices),  [modeling](https://2020.igem.org/Team%3ATU_Darmstadt/Model) and the theoretical planning of our project. Furthermore, we started the search for [sponsors](https://2020.igem.org/Team%3ATU_Darmstadt/People/Sponsoring) for the upcoming costs.

Week 13

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.

## April

Week 14

During our research we came up with the idea of **[enzymes](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation) in the [biofilm](https://2020.igem.org/Team%3ATU_Darmstadt/Project/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](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Degradationofsubstances) to degrade the antibiotic azithromycin**.

Our brainstorming about ideas to get in contact with society resulted in the first idea to **make a [podcast](https://2020.igem.org/Team%3ATU_Darmstadt/Education#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.

Week 15

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**.

Week 16

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**.

Week 17

This week we started **contacting a couple of [experts](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices)**, 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](https://2020.igem.org/Team%3ATU_Darmstadt/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](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Experimentalapproach) as possible for a proof of concept in *E. coli*.

Week 18

**Our first modeling approach was to create a ligand that [docks to laccase](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#Docking) and diclofenac**, because according to our research and our **first expert talk with [Udo Bäuerle](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Implementation)** 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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Ethics) 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.

## May

Week 19

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.

Week 20

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](https://2020.igem.org/Team%3ATU_Darmstadt/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](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#iJET) where we wanted to send a paper plane around the world to connect all teams**. Additionally, **another [collaboration about modeling with Hannover](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#HannDa)collaboration 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](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#Meetups) 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.

Week 21

This week **we optimized the [docking for the laccase modeling](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#Docking)** 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](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm#Displaying_Enzymes)** (an extracellular matrix protein of the biofilm). The expert **Prof. Dr. Susanne Lackner from TU Darmstadt told us about [floating bodies](https://2020.igem.org/Team%3ATU_Darmstadt/Implementation#Wastewater%20Treatment%20Plants) 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**.

Week 22

Our project now took on more shape, as we decided on two [suitable laccases](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Degradationofsubstances), 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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#EreB_CM)**. Therefore, as a **collaboration with Hannover we thought about a [biofilm modeling](https://2020.igem.org/Team%3ATU_Darmstadt/Model/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.

## June

Week 23

The iGEM Team Duesseldorf hosted as every year a **[postcard collabo](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#Postcards)** 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.

Week 24

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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Implementation) from the wastewater treatment plant in Weiterstadt**.

Week 25

**We talked to [Dr. Christian Dietz](https://2020.igem.org/Team%3ATU_Darmstadt/Attributions) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Synthetic_Biology) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Project/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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#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](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)**. Luckily, we found the iGEM team Stuttgart which project is very close to ours.

Week 26

The ethical considerations for our project resulted in the aim to **make a [guidance for the correct use of our project](https://2020.igem.org/Team%3ATU_Darmstadt/Safety/How_To_Handle_B-Tox)** 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](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)**. Kaiserslautern also invited us to the [Wastewater group meeting](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#WWEurope), 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)](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#EreB_MD) and also compared our laccase structures with the laccase structure of *Trametes versicolor*.

## July

Week 27

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](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Degradationofsubstances). To simulate the water flow of the wastewater treatment plant, **we would like to build a [flow chamber](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm#Flowchamber) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#EreB_MD).

Together **with Kaiserslautern and Stuttgart we planned to collect all wastewater treatment information [on one page](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)** 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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#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.

Week 28

For the [toxicity assay](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Experimentalapproach) 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.

Week 29

To **help other iGEM Teams to [build their own flow chamber we made DIY modules](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution)**. 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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Environment)**. [Patrick Schröder](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Environment) from the Federal Environment Agency in Germany, talk with us about the [appropriate disposal of medication](https://static.igem.org/mediawiki/2020/9/9d/T--TU_Darmstadt--How_to_properly_dispose_of_pharmaceutical_products.pdf). 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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling) started in MATLAB**. We continued to model the EreB docking with azythromycin and worked on an easier way to evaluate our modeling results.

Week 30

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**.

Week 31

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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#Livestream) with Kaiserslautern and our risk workshop took place this week**.

To integrate the [kill switch](https://2020.igem.org/Team%3ATU_Darmstadt/Project/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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling) has been created.

## August

Week 32

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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Synthetic_Biology)** about microbial genomics and biofilm formation.
We got the results from the interview from Kaiserslautern with [Dr. Ulrich Ehlers](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Implementation) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Implementation#GMOs%20in%20Wastewater%20Treatment). 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**.

Week 33

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](https://2020.igem.org/Team%3ATU_Darmstadt/Safety)**. 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.

Week 34

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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Biofilm_Modeling) 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.

Week 35

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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Implementation) 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.

## September

Week 36

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](https://static.igem.org/mediawiki/2020/d/dc/T--TU_Darmstadt--CheckIn_Form_EreB_iGEM20.pdf)**. 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.

Week 37

This week we decided which [awards](https://2020.igem.org/Team%3ATU_Darmstadt/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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#School_Visits)**. 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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling#fus) *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.

Week 38

The print of the flow chamber goes on and the **fourth [podcast](https://2020.igem.org/Team%3ATU_Darmstadt/Education#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](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Biofilm_Modeling) grow under defined conditions.

Week 39

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](https://2020.igem.org/Team%3ATU_Darmstadt/Safety) 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.

Week 40

We had a **talk with [Prof. Dr. Alfred Nordmann](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Ethics) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution#Flow%20Chamber) 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.

## October

Week 41

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**.

Week 42

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](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Synthetic_Biology) to our questions. Furthermore **we talked with [Prof. Dr. Ralf Möller](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Implementation) 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.

Week 43

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](https://2020.igem.org/Team%3ATU_Darmstadt/Education#School_Visits) 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](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations#Highlight%20the%20sustainable%20development%20impact)**, to show how our work contributes to the [sustainable development goals](https://2020.igem.org/Team%3ATU_Darmstadt/Sustainable) of the United Nations.

In the next weeks until the jamboree we will finish the wiki, the presentation video and the poster. In addition we will prepare for the final jamboree and judges questions.

Thank you for following our journey.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TU Darmstadt/Engineering

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O

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[![logo](https://static.igem.org/mediawiki/2020/4/47/T--TU_Darmstadt--black_owl.png)](https://2020.igem.org/Team%3ATU_Darmstadt)

* PROJECT

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Description)[BACKGROUND](https://2020.igem.org/Team%3ATU_Darmstadt/Background)
  [IMPLEMENTATION](https://2020.igem.org/Team%3ATU_Darmstadt/Implementation)[PHARMACEUTICAL DEGRADATION](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation)
  [BIOFILM](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm)
  [KILL SWITCH](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Kill_Switch)[NOTEBOOK](https://2020.igem.org/Team%3ATU_Darmstadt/Notebook)
  [EXPERIMENTS](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments)
  [BACKBONES](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Backbones)
* PEOPLE

  [TEAM](https://2020.igem.org/Team%3ATU_Darmstadt/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Attributions)
  [COLLABORATIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations)
  [SPONSORING](https://2020.igem.org/Team%3ATU_Darmstadt/People/Sponsoring)
* MODELING

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Model)[ENZYME MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling)
  [BIOFILM MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Biofilm_Modeling)
  [KILL SWITCH MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling)[ROSETTA GUIDE](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Rosetta_Guide)
* HUMAN PRACTICES

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Human_Practices)[INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices)
  [SCIENCE COMMUNICATION & EDUCATION](https://2020.igem.org/Team%3ATU_Darmstadt/Education)
  [RESPONSIBLE RESEARCH](https://2020.igem.org/Team%3ATU_Darmstadt/HP/Ethics)
* PARTNERSHIP

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)[SUMMARY & METHODS](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Summary_And_Methods)
  [EXPERTS & LITERATURE](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Experts_And_Literature)
* SAFETY

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Safety)[HOW TO HANDLE B-TOX](https://2020.igem.org/Team%3ATU_Darmstadt/Safety/How_To_Handle_B-Tox)
* ACHIEVEMENTS

  [AWARDS](https://2020.igem.org/Team%3ATU_Darmstadt/Awards)[BEST SOFTWARE](https://2020.igem.org/Team%3ATU_Darmstadt/Software)
  [BEST SUSTAINABLE DEVELOPMENT IMPACT](https://2020.igem.org/Team%3ATU_Darmstadt/Sustainable)[PARTS](https://2020.igem.org/Team%3ATU_Darmstadt/Parts)
  [CONTRIBUTION](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ATU_Darmstadt/Engineering)
* JUDGING FORM

  [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3429)[POSTER](https://2020.igem.org/Team%3ATU_Darmstadt/Poster)

# Engineering Success

[Degradation Enzymes](#Degradation_Enzymes)
[Displaying our Enzymes in the Biofilm Matrix](#Displaying_Enzymes)
[Bacterial On-/Off-switch - How to contain B-TOX](#Kill switch B.Sub)

[Degradation Enzymes](#Degradation_Enzymes)
[Displaying our Enzymes in the Biofilm Matrix](#Displaying_Enzymes)
[Bacterial On-/Off-switch - How to contain B-TOX](#Kill switch B.Sub)

Although we weren’t able to go into the lab this year, due to the current COVID-19 pandemic, we **planned our laboratory work in detail**. Our aim was to **learn about typical problems** in the lab and **how to solve them**. Following the **design-build-test engineering cycle**, we came up with a plan how to evaluate our experimental outcome and deal with any unexpected results.

## Degrading Diclofenac and Azithromycin by using Degradation Enzymes - Laccase and EreB

As we seek to degrade specific micropollutants in wastewater, we chose **two bacterial laccases and one esterase** to work with. Laccases are able to oxidize phenolic compounds, such as diclofenac, and the chosen esterase, EreB, is able to degrade erythromycin and also shows activity towards azithromycin, which is similar in structure. We want to express the three enzymes in *E. coli* strains with a strong promoter for high protein production rates. Cloning will be verified via (commercial) **Sequencing**. Purification of the target protein will be ensured by a **fused StrepTag II affinity tag**. After performing an SDS-PAGE analysis, a large **band is expected at the protein size** of the expressed enzyme (CueO: 57.81 kDA, CotA: 59.74 kDa, EreB: 49.21 kDa), since the enzymes are connected to a **strong T7-promoter. Important kinetic data of our transformation enzymes** can be measured after successful protein expression and purification.

Laccases are **multicopper enzymes** belonging to the group of oxidases that **catalyse the oxidation of phenolic units**  in lignin as well as a wide variety of organic compounds including various types of phenols and aromatic amines. Due to this oxidoreductase activity laccases can react with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) also known as **ABTS** as a substrate (figure 1). We would have used ABTS in a **spectrophotometric assay** to determine the activity of our laccases. This assay is based on the formation of a stable radical cation by enzymatic

[![figure](https://static.igem.org/mediawiki/2020/c/cc/T--TU_Darmstadt--ABTS.png)](https://static.igem.org/mediawiki/2020/c/cc/T--TU_Darmstadt--ABTS.png)

**Figure 1:** Structure of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

oxidation. The resulting cation can be measured photometrically[[1,](#cite_note-1)[2]](#cite_note-2). Accordingly, the laccase activity would have been calculated from the **optical density (OD) changes at linear range with 420 nm**.
We would have performed the [enzyme assay](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments) immediately at 30 °C after mixing the enzyme solution and substrate buffer (phosphate-citrate-buffer, pH 4.0). For the calculation of the enzyme activity we would have defined the transformation of 1 µmol substrate to product per minute as one unit of enzyme activity[[2]](#cite_note-2).

On the one hand, the gathered data could confirm the **successful production of active enzyme** and thus serves as a **quality control**. On the other hand, we would have a **defined enzymatic activity of our produced laccases** and could **compare it to the activity of transformation of pharmaceutical compounds like diclofenac** to evaluate a kind of efficiency. If we would not have been able to detect any enzymatic activity there is most likely a problem with the production or storage of our enzymes. Maybe the recombinant production of our laccase did not work properly, and only inactive forms were produced due to protein folding problems. In this case we would have to rethink our production process and change it accordingly.

As this assay only accounts for the laccases, we would have performed a different activity assay for EreB. Prior to EreB site saturation mutagenesis, a simple **[Kirby Bauer assay](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments) will be performed to roughly illustrate the activity of EreB** for both erythromycin and azithromycin. Note that the results are relative, therefore the experiment would have been carried out **in close proximity to the approach of [iGEM Munich 2013](https://2013.igem.org/Team%3ATU-Munich)**. For that, the same reporter organism, which would be *Micrococcus luteus*, would need to be grown on an agar plate. Also note that the experiment would need to be **repeated after mutagenesis of EreB**, as the test has no absolute outcome and instead provides results for a qualitative comparison. This relative comparison prior/after mutagenesis can **demonstrate improvements in azithromycin degradation ability**. The best outcome would be decreased sizes of inhibitory zones, as that would mean **increased degradation activity** for the same amount of time. To preserve the amount of enzyme available, a general, qualitative-only incubation of EreB with erythromycin and azithromycin would have been performed with a negative control group, as no positive control is available for this enzyme yet. For this we would have incubated an equimolar amount of erythromycin and azithromycin respectively onto two filter plates, one containing EreB and one containing buffer only. With standard *E. coli* strains or *Micrococcus luteus*, we would have **checked inhibitory effects of erythromycin and azithromycin**. If the negative controls showed significant inhibitory zones while the second plate did not, we could qualitatively prove the degradation capability of EreB[[3,](#cite_note-3)[4]](#cite_note-4). The exact concentration necessary for EreB to perform this assay can only be determined in the lab, as no literature is available. The concentration in the table listed below can be a good starting point and was used in a comparable assay from iGEM Munich 2013[[3]](#cite_note-3):

| Substance | Amount | Stock solution |
| --- | --- | --- |
| EreB recombinant protein 40 nM | 0.18 µl | 11 µM EreB in PBS with 10 mM ß-mercaptoethanol, 2% glycerol (v/v) and 300 mM NaCl |
| Erythromycin 0.36 mM or Azithromycin 0,36 mM | 3 µl | Erythromycin/Azithromycin in ethanol, 6 mM / 6 mM |
| Tris-HCl buffer pH 7.5 100 mM | 10 µl | Tris-HCl buffer pH 7.5, 500 mM |
| NaCl 0.08 M | 4 µl | NaCl in water, 1 M |
| ddH20 | 32.82 µl | ddH20 |
| **TOTAL:** | **50 µl** |  |
Table 1: Suggested concentrations of protein EreB and its substrates azithromycin and erythromycin for the Kirby-Bauer-Assay as used by iGEM Munich 2013[[3]](#cite_note-3)

To solve the various problems of our project we need appropriate and robust analytical methods. One of the main topics of our project is to **enable and improve the degradation capabilities** of diclofenac by the bacterial laccases CueO ([BBa\_K3429012](http://parts.igem.org/Part%3ABBa_K3429012)) and CotA ([BBa\_K3429011](http://parts.igem.org/Part%3ABBa_K3429011)), as well as improving the degradation of azithromycin by EreB ([BBa\_K1159000](http://parts.igem.org/Part%3ABBa_K1159000)), as the latter is already able to degrade the structurally similar erythromycin. We therefore chose **High Performance Liquid Chromatography** (HPLC) as a powerful and robust analytical method. HPLC analysis would have enabled an evaluation as well as a comparison of the degradability. As for all three enzymes, we would thus **determine the enzymatic degradation and degradation rate** of the respective wastewater polluting drugs. Moreover, we could verify the educt transformation **with HPLC analysis**.

The detection method is based on the **UV-absorbance of the substrate** after its retention by a modified porous silica material. As for the laccases, we would have analysed a **commercially available standard of the target substance diclofenac** (Merck, Darmstadt, #38429)[[5]](#cite_note-5). Thereby, we would have determined the **method specific retention time** and the **UV-absorbance at different concentrations**. Based on this initial test, we could analyse and estimate the substrate conversion in the reaction mixture containing our laccase in comparison to a reaction mixture lacking the laccase. On the one hand we could have observed the **decrease of absorbance from diclofenac in presence of the laccase**, which indicates its functionality. In this case, new absorbance signals according to the transformation products should be detectable. Based on this data we would have been able to estimate a degradation rate of the substance by the used laccase, which in turn would have allowed us to compare those degradation rates for the different enzymes and their modifications. This would greatly help to identify beneficial enzyme **modifications**.

On the other hand, we could have observed **no significant change in the absorbance signal**. In this regard, ”significant” would be a change **clearly outside the defined error limits determined by the standard**. No significant change would mean that no enzymatic degradation of the substrate has taken place. In this case, we would have gone through the whole assay step by step to **identify possible sources of error**. A possible problem might be inactive enzyme. Then we would have to determine the enzymatic activity by the ABTS assay again. When no error could have been found then maybe our enzyme could not have been able to degrade the substrate. Then we should try a different mutation approach or use another enzyme.
As we would have performed the same HPLC kinetic activity assay with EreB as we did for the Laccase, the discussion presented above can also be applied. The best outcome of the enzymatic assays for EreB would be an **increased activity of EreB regarding azithromycin**, and ideally, a constant or improved activity regarding erythromycin. This implies that enzymatic activity assays must be **performed at least twice**, prior and after site saturation mutagenesis, for both erythromycin and azithromycin.

In addition to HPLC analysis, we planned to do a **LC-MS** to identify the erythromycin and azithromycin **degradation products**. As the mechanism of reaction starts with the cleavage of the macrolide ring, in sequence to which the added water molecule is eliminated during the acetal formation, the **substrate and the product show identical molecular mass**. We must therefore detect and interpret the **open lactone intermediate** which is expected to be 18 g/mol heavier due to H2O addition. The molecular mass of erythromycin is 733,5 g/mol, while the hydrolyzed hemiketal is 751,6 g/mol[[6]](#cite_note-6). For azithromycin the respective molecular masses are 748,98 g/mol for the macrolide and 768,98 g/mol for its degradation product. Unless the antibiotics are fully degraded during incubation, the LC is expected to yield three successive peaks with partly different molecular masses and retention times for the cleaved and the non-degraded molecules. It is therefore possible, through comparison of the calculated to the measured molecular weight, to **determine whether the molecule is cleaved by EreB** the way it is expected from literature. The exact protocol for the suggested LC-MS gradient can be found on our [experiments page](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments). As the product does not differ from the intermediate substantially in terms of molecular weight, the gradient of water and formic acid used might need adjustments until the peaks are clearly differentiable based on their retention times. We expect to see a single peak in the control group with untreated erythromycin or azithromycin, and three peaks for the samples containing EreB, of which the molecular mass of the substrate and the final product vary only in their retention times and are otherwise indistinguishable during LC.
By site directed mutagenesis we hope to **achieve** an increased affinity of our laccase-oxireductases towards diclofenac (figure 2). We chose the targeted amino acids based **on already published discussions for directed mutagenesis** on the corresponding enzymes. Further experiments following our laccase-activity assays are needed to **quantify the increase/decrease in activity**. An ABTS assay is appliable for measurement of unspecific transformation activity and HPLC analysis for target specific measurements of substrates that do not absorb visible light spectra. The same conditions as with the wildtype enzymes should be used here to increase comparability.

[![](https://static.igem.org/mediawiki/2020/d/d1/T--TU_Darmstadt--SDM.svg)](https://static.igem.org/mediawiki/2020/d/d1/T--TU_Darmstadt--SDM.svg)

**Figure 2:** Site directed mutagenesis. The plasmid containing the GOI is replicated using a primer pair with a built in mutation to replicate nicked plasmids containging the desired mutations. The plasmids are then transformed into competent cells for ligation and expression of the engineered protein.

Not only **positive mutants showing an increase in activity should be used in further experiments** but also primary lower efficiency laccase variants, as there might be hidden epistase effects (the mutation at one site can influence the effect of other mutations). This way even unfavorable seeming mutants can cause an increase in enzymatic activity by changing the impact of other mutations by specific epistasis. Therefore, mutation sites should also be combined to check their compatibility and mutual effects. Experimental results can be predicted/verified by small molecule ligand docking.

Since there are no publications on directed mutagenesis of EreB, we consider a **site saturation mutagenesis (SSM) approach**. Because wild type EreB already shows **promiscuous activity** to azithromycin, we hope to improve the enzyme to bind and degrade azithromycin more effectively. As shown before, enzymes with promiscuous activities towards other substrates are a great starting point for the directed evolution of enzymes for new substrate specificities. For SSM we want to focus on the residues close to EreB’s active site to **improve substrate affinity and accessibility**. Mutants can be screened by either Kirby Bauer Assay or HPLC analysis. Unfortunately both assays are no methods capable of high throughput screening for positive mutants so we want to focus on mutating residues close to EreB's active site to generate a limited, screenable amount of mutants for **low-throughput screening** towards improved activity on azithromycin.

The Zebrafish Embryo Acute Toxicity Assay would have been used to **determine the acute fish toxicity** as well as the **developmental and reproductive toxicity** (DART) of the enzymatic transformation products from wastewater polluting drugs such as diclofenac and the antibiotic azithromycin. The comparison of the enzymatic transformation products with the original substance provides an **evaluation of whether the laccase/EreB treatment reduces the toxic impact of these drugs on the aquatic environment**.
For this purpose, embryos of the zebrafish (Danio rerio) are used, which are an **established model in ecotoxicology** and a good **alternative to animal experiments** on adult fish species (figure 3). This assay is based on the OECD Test Guideline 236 for the Fish Embryo Acute Toxicity (FET) test and is used to **determine the lethal concentration LC50 value of diclofenac, azithromycin and their transformation products**. The calculation of the LC50 value is based on four apical observations as indicators of lethality within a period of 96 hours post fertilization.

[![figure](https://static.igem.org/mediawiki/2020/f/f6/T--TU_Darmstadt--FischiesmögenDiclonicht.png)](https://static.igem.org/mediawiki/2020/f/f6/T--TU_Darmstadt--Fischiesm%C3%B6genDiclonicht.png)

**Figure 3:** By using a Zebrafish Embryo Acute Toxicity Assay, we efficiently prove the decrease of toxicity for aquatic environment. The assay allows measurement of toxicity to a tailored model-organism, without doing actual animal testing.

Using negative controls consisting of a water and a solvent control as well as a positive control with the substance 3,4-dichloroaniline we would have gotten **defined limits**.
In addition, we would have tested the initial substance. With this experimental setup we would have been able to **compare the caused toxicity of the enzymatic transformation products with the toxicity of the initial substance**.
A positive outcome would have been that the transformation product is less toxic than the initial substance or does not show any toxicity at all, by comparison with the solvent control. If enzymatic degradation has been occurred but the transformation products had the same toxic effect as the initial substance, we would have used those transformation products in a new experiment to show the biodegradability within bioactive sludge. It could have been that the transformation products are still toxic but show better biodegradability and could therefore be better removed in sewage treatment plants.
This method would have enabled us to **observe a direct effect of performed enzyme modification** to the resulting degradation ability. By taking this into account we could have identified enzyme variants for further optimization and fine-tuning.

[![DCF_Tranfo](https://static.igem.org/mediawiki/2020/7/79/T--TU_Darmstadt--DCF_Transformationsprodukte_ToxAssay.png)](https://static.igem.org/mediawiki/2020/7/79/T--TU_Darmstadt--DCF_Transformationsprodukte_ToxAssay.png)

**Figure 4:** Diclofenac transformation products. Confirmed enzymatic transformation products of diclofenac by the laccase from *T. Versicolor*.

Like the laccases CotA/CueO, EreB is intended to be **surface displayed in the matrix of *B. subtilis***. To guarantee its functionality, in vitro assays should be under oxidizing conditions (air) as they are present in the biofilm environment. The display itself can only be tested in vivo (correct biofilm-formation and orientation of the fusion protein can hardly be predicted). It is **expected, that *B. subtilis* can appropriately fold EreB, CueO and CotA**.

Both for *E. coli* and *B. subtilis*, in vivo assays on survival in erythromycin/azithromycin or diclofenac contaminated environments would be of interest. However, the low concentrations of erythromycin/azithromycin or diclofenac in wastewater are not expected to inhibit bacterial growth, and measurement of susceptibility of bacteria for higher pharmaceutics concentration is not representative for the final application. The impact of exceptionally low concentrations is hardly measurable in in vivo assays.

For *B. subtilis* expression, the plasmid encoding EreB and CueO were also codon optimized within the sequence (CotA is orginated from *B. subtilis*), however this would require an additional cloning step.

## Displaying our degradation Enzymes in the Biofilm Matrix - Fusion Proteins with TasA

Based on our research our goal was to [**degrade toxic compounds in wastewater by enzymes**](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation). These enzymes should be stable and work as efficient as possible. Therefore, we imagined different solutions. Besides secreting our enzymes by microorganisms into the wastewater or add purified enzymes, immobilization would be a possible solution. Immobilization can be realized on various materials but also by using biological systems.  **Huang et al. reached immobilization of proteins and enzymes in the biofilm matrix of *Bacillus subtilis***[[7]](#cite_note-7). They successfully built fusion proteins of the matrix protein TasA with other proteins or enzymes[[7]](#cite_note-7).
Considering the manifold advantages of [immobilization](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#Enzyme_Immobilization) and the research of Huang et *al.*  we decided to display our degradation enzymes in the biofilm matrix of *B.  subtilis* as [**fusion proteins with TasA**](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm#Displaying_Enzymes). The matrix protein TasA fits best as a fusion protein because it is a main compound of the biofilm matrix leading to a high number of enzymes displayed. By adjusting the fusion proteins, we could **display different enzymes**, making our project more modular. Since we use a living system, we avoid elaborate purification of enzymes and can easily adapt our biofilm.

Huang et al. already showed that **fusion proteins consisting of TasA and another protein** (e.g. mCherry or SpyTag proteins) are **successfully exported into the biofilm matrix**. Even a fusion protein of TasA with the enzyme MHETase (63.1 kDa), that is bigger than the enzymes we want to use, was localized in the biofilm matrix of *B. subtilis*.
We **conceptualized fusion proteins with our degradation enzymes and the protein TasA** ([BBa\_K3429001](http://parts.igem.org/Part%3ABBa_K3429001)) based on the research of Huang et *al*  The genes of our laccases CotA and CuO are fused to the 3' end of the *tasA* gene with a gene fragment encoding a **flexible glycine-serine linker** (ARGGGGSGGGGS)([BBa\_K3429003](http://parts.igem.org/Part%3ABBa_K3429003)), that was also used by Huang et *al.*

[![figure](https://static.igem.org/mediawiki/2020/b/b0/T--TU_Darmstadt--Engineering_Success_TasA.svg)](https://static.igem.org/mediawiki/2020/b/b0/T--TU_Darmstadt--Engineering_Success_TasA.svg)

**Figure 5:** The degradation enzymes are displayed in the biofilm matrix as fusion proteins with the matrixprotein TasA. The gene for the fusion protein is integrated into the genome of *B. subtilis*. TasA is fused to the enzyme via a flexible glycine-serine linker.

The **fusion protein will be inserted into an appropriate plasmid via Gibson Assembly**. Overhangs and primers were **designed successfully *in silico*** with the software SnapGene. As a plasmid we use the **shuttle vector [pSEVA3b67Rb](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Backbones)**[[8]](#cite_note-8). This high copy plasmid is compatible as reporter for *Escherichia coli* and *B.  subtilis* and has a chloramphenicol resistance[[8]](#cite_note-8). This resistance will be exchanged against an ampicillin resistance via Gibson Assembly, because the strain we would like to use for our proof of concept experiments also owns a chloramphenicol resistance.
Cells of the ***B.  subtilis* strain GP1622** will be transformed with this plasmid using a [protocol](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments) obtained from [Prof. Stülke](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices#Jörg_Stülke) (Georg-August-Universität Göttingen)[[9]](#cite_note-9). The strain also obtained from Prof. Stülke **lacks the genes *sinR* and *tasA***[[9]](#cite_note-9). With the *sinR* knockout we [**improve the biofilm formation**](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm#Improvement_Biofilm) and the [*tasA* knockout](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm#TasA_knockout) makes sure that **only our fusion proteins are integrated into the biofilm matrix**[[10]](#cite_note-10).
As a **proof of concept**, we built a fusion protein of **TasA and superfolder green fluorescent protein (sfGFP)**  ([BBa\_K3429002](http://parts.igem.org/Part%3ABBa_K3429002)). The sequence of the *sfGFP* gene is codon optimized for *B.  subtilis* and fused to *tasA* as described above. The display of the TasA-sfGFP fusion protein in the biofilm matrix can be **verified using fluorescence microscopy**. Therefore, we would use the measurement kit of iGEM to standardize our results.
We hypothesize that TasA fusion proteins with our targeted degradation enzymes will succeed, if TasA-sfGFP is successfully expressed in the biofilm matrix.

In the following we **discuss possible outcomes and possible adaptions** of our experimental setup.
If our biofilm is built correctly but we are unable to measure any fluorescence we can conclude that there has to be a problem with the sfGFP. It can mean that it was not even expressed or lost its function due to the fusion with TasA. To react on the former, we would **check the sequences** of our ordered sfGFP-TasA gblock as well as doing a **plasmid preparation with following sequencing** of our PCR amplified fusion protein parts. Approaching to a possible loss of function due to the fusion with TasA we would try to use a **different linker**. A potential reason for the loss of function can be too less space between the proteins, leading to misfolding or aggregation. This can be solved with a **longer and more rigid linker**[[11]](#cite_note-11). Another reason could be that sfGFP cannot be folded fast enough before secretion into the matrix with TasA. This can also be adapted by changes in the linker sequence[[12]](#cite_note-12). We would try to use **amino acids that slow down the translation** of the fusion protein, for example proline. By **testing different protein architectures**, we can learn more about the TasA-fusion platform in general, **helping other researchers** who also want to use this technology.
If the biofilm we grow seems to be less stable and we are able to detect fluorescence in our cells the export of TasA-sfGFP into the biofilm matrix is detracted by the fusion of TasA to sfGFP. In this case we would redesign our fusion protein especially regarding to the used linker as well as **checking the secretion sequence** for the transport of TasA.
A less stable biofilm combined with lack of any fluorescence could be explained with an unsuccessful transformation of our *B. subtilis* cells, a mistake in the sequence of our fusion protein or a mistake in our plasmid. Promoter, ribosome binding site or selection marker could be defective.
**Further steps** would be the repetition of the described approach with **TasA-CueO**, **TasA-CotA** and **TasA-EreB fusion proteins**. The successful display of these enzymes in the biofilm matrix could be verified using our flow chamber and the [ABTS assay](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation#ABTS). We already [**modeled**](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling) our **new composite part**, the TasA-EreB fusion protein ([BBa\_K3429013](http://parts.igem.org/Part%3ABBa_K3429013)) and the TasA-CotA fusion protein, via Molecular-Dynamic simulation. Furthermore, for the implementation of our biofilm the gene for our **fusion proteins** should be **integrated into the genome** of the *B. subtilis* strain via **homologous recombination** using integrative vectors[[13]](#cite_note-13).

## Bacterial On-/Off-switch - How to contain B-TOX

As already previously described, this year's iGEM project is a solely theoretical project **due to the COVID-19 pandemic**. Therefore, the experimental setup was only theoretically designed, and we **elaborate some expected results based on literature references**.

The [**Cre recombinase**](http://parts.igem.org/Part%3ABBa_K1680007) allows the **inversion** of the DNA region between its two **head-to-head aligned** [**recognition sites lox 66**](http://parts.igem.org/Part%3ABBa_I718016) [**& 71**](http://parts.igem.org/Part%3ABBa_I718017), and was previously used by the [iGEM team of Tuebingen 2015](https://2015.igem.org/Team%3ATuebingen) [[14]](#cite_note-14). For our [kill switch](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Kill_Switch#FinalKillSwitch), we use the Cre recombinase to inverse the promoter region **controlling the expression of an essential gene** in *B. subtilis*. The expression of the **genomically integrated Cre recombinase cassette** is controlled by the xylose-inducible promoter [**PXylA**](http://parts.igem.org/Part%3ABBa_K733002). As the recombinase has a very important function in our kill switch, we first aim to test the recombinase activity and the correctness of our cassette design.

#### Assaying single inversion of our promoter region by Cre recombinase:

For testing the Cre recombinase activity in *B. subtilis*, we use a plasmid designed by [iGEM Team Paris 2007](https://2007.igem.org/Paris) with the lox sites 66 & 71 aligned head-to-head [[15]](#cite_note-15). Between the two recombinase sites the **constitutive promoter Pveg** is located on the **antisense strand**, but if the DNA region is inversed, Pveg controls the expression of a **reporter gene for quantification** of protein production, *e.g.* **green fluorescent protein (GFP)**. Figure 6 shows the schematic layout of the cassette for testing Cre recombinase activity.
[![DCF_Tranfo](https://static.igem.org/mediawiki/2020/1/10/T--TU_Darmstadt--Engineering_successGFP.png)](https://static.igem.org/mediawiki/2020/1/10/T--TU_Darmstadt--Engineering_successGFP.png)

**Figure 6:** Schematic representation of the designed GFP expression cassette to assay recombinase activity and genomically integrated the recombinase expression cassette. GFP expression is under the control of a upstream DNA region(red) flanked by the two recombinase sites (lox 66 and 71, grey arrows) aligned head-to-head. The sense strand of the DNA region controlling GFP is empty, but the antisense strand encodes the constitutive promoter Pveg. The addition of xylose induces the expression of the Cre recombinase. As a result, the DNA region is inversed and the constitutive promoter Pveg starts expressing GFP as a quantifiable signal.

The cassette is cloned into a **replicative plasmid with a selection marker**, and the plasmid is transformed into a ***B. subtilis* strain** containing a **genomically integrated Cre recombinase expression cassette**. As a control, we use the same ***B. subtilis* strain not harboring the plasmid** in the following experiment. The test culture and the control culture are **supplemented with xylose** in the exponential growth phase to induce Cre recombinase expression. If the **Cre recombinase is active** and the system correctly designed, Cre recombinase **inverses the promoter region on the plasmid a single time**. Consequently, the reporter gene encoding **GFP** is expressed by the **constitutive promoter Pveg** and the GFP signal can be  **qualitatively determined by visual observation or quantitatively by fluorescence spectroscopy measurement**. In the **control culture no GFP expression is expected** and it can therefore be used as negative control for GFP quantification. This experimental setup additionally allows the testing of **necessary inducer concentrations** to achieve **sufficient expression levels** and enables the investigation of further parameters to optimize the system, *e.g.* media composition or cell density.

#### Assaying multiple inversion of our promoter region by Cre recombinase:

In the literature, the **potential double inversion** between the lox sites is, to our knowledge, **not described**. Among others, the iGEM Team Paris 2007 characterized the **lox site 66 and 71** as sites for **"irreversible" rotation**[[15]](#cite_note-15). Nevertheless, we want to assure that **double inversions are not possible**. For this test the cassette described in figure 6 needs to be slightly modified by **fusing a C-terminal SsrA degradation tag** to the reporter protein[[16]](#cite_note-16). This is necessary as GFP has a **half-life period** which is **too long** to be used within our developed assay, but the **fused degradation tag** leads to a **faster degradation** of the tagged protein in the cell[[16,](#cite_note-16) [17]](#cite_note-17). The **procedure is initially identical** to the procedure described above for **assaying single turns** by Cre recombinase. The plasmid is transformed, the cells are allowed to grow and the expression of the Cre recombinase is induced by adding xylose in the exponential growth phase. As already described, a GFP signal is expected, which indicates a single inversion of the cassette. The **xylose-containing media** is subsequently exchanged to **fresh media** with or without supplemented xylose. In the **absence of xylose**, Cre recombinase expression stops and **recombinase activity shrinks over time**. Consequently, in the absence of xylose another **inversion is less likely due low recombinase activity**. In the **presence of xylose**, the Cre recombinase is further on expressed and a **second inversion may occur**, if Cre recombinase is capable of its catalysis. Consequently, if the Cre recombinase is able to inverse the promoter more than once, the GFP expression in presence of xylose is expected to shrink over time. Addition of the degradation tag shall help to decrease the incubation times in this assay.

This setup only allows a qualitative assay. For a quantitative measurement, the sequencing of single colonies is likely inevitable[[18]](#cite_note-18).

#### Assaying functionality of the kill switch design:

When eventually testing our final kill switch design, it is **important to quantify the deadliness** of our kill switch. We have to ensure that our kill switch does what it is supposed to do: Killing our genetically modified *Bacillus subtilis* cells upon leaving the biofilm environment. It is well known that, some **antibiotics** are able to **kill cells by blocking the activity of protein biosynthesis or ribosomal proteins**, but we don't know if this also **applies to our [kill switch model](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling)** [[19,](#cite_note-19) [20,](#cite_note-20) [21]](#cite_note-21).

For this purpose, we **compare the growth behavior** of a *B. subtilis* strain harboring the genomically integrated [kill switch](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Kill_Switch#FinalKillSwitch) (figure 7) with a control strain not harboring the kill switch cassette.
![DCF_Tranfo](https://static.igem.org/mediawiki/2020/9/9c/T--TU_Darmstadt--Switchies_Evolution_final_cassette.png)

**Figure 7**: Schematic representation of the designed final kill switch cassette. RpsB expression is under the control of a upstream DNA region (red) flanked by the two recombinase sites (lox 66 and 71, grey arrows) aligned head-to-head. The sense strand encodes the constitutive promoter Pveg and the antisense strand encodes the inducible PdegQ. The addition of xylose induces the expression of the Cre recombinase. As a result, the DNA region is inversed and the inducible PdegQ starts expressing rpsB.

Both cultures are grown to a **defined cell density** until the xylose is added to **induce expression** the Cre recombinase cassette. This will cause the **inversion of the kill switch cassette**, as described above and the expression of *rpsB* will then be under the **control of the cell density sensitive degQ promoter PdegQ**. (figure 7 below ) Both cultures are then **strongly diluted with fresh medium** and the cells are incubate to grow. The **growth behaviour** of both cultures can be determined by measuring the **cells absorbance at 600 nm (OD600) at defined time points**. In addition, we will perform **live/dead staining** with SYTO 9 or propidium iodide stain to compare the **ratio of dead to alive bacteria** in the kill switch-harboring strain and the control strain[[22]](#cite_note-22). We expect that the **growth curves** of the two strains will **differ significantly**, if the kill switch design is functional. The strain with the **established kill switch** should **not grow** after dilution as the quorum sensing signal is not strong enough to induce expression of *rpsB*. Ideally, the majority of the cells of this strain are dead to confirm **high deadliness of the kill switch mechanism**. Using the experimental setup, it is also possible to **determine the necessary cell density to activate the PdegQ** by diluting the bacteria in various ratios. Cultures not passing the threshold will show bacterial growth similar to the control. However, the functionality of the degQ promoter can also be verified by another method.

#### Assaying the degQ promoter behavior :

For this alternative assay, the kill switch-harboring cells are grown in separate test flasks up to a defined cell density and recombinase expression is induced. We hypothesize that **if the cell density is not high enough to trigger a quorum sensing signal, the induced inversion of the promotor region leads to the death of the cells**. If the cells however **continue growing**, the **density during induction is high enough**. As positive growth control a *B. subtilis* strain not harboring the kill switch is tested. Cell viability and consequently the **expression of the essential gene** can be determined by **live/dead staining**, as already described above.

#### Assaying the Pveg promoter strength :

In our kill switch design, we use a **different constitutive promoter Pveg** than the **naturally encoded** promoter controlling *rpsB* expression in *B. subtilis*. Consequently, it is possible that the used **promoter is stronger** than the original and **RpsB is overexpressed**. We want to test whether 1) the ***rpsB* expression** controlled by Pveg **differs from the original expression level**, and 2) whether a shifted level has an **influence on the *B. subtilis* fitness**. For this purpose, we grow our *B. subtilis* strain haboring the established kill switch cassette and a strain not harboring the cassette to a **certain cell density** and then compare the expression level of RpsB by reverse transcriptase qPCR[[23]](#cite_note-23). A qPCR assay templated by the reverse transcripted *rpsB*-encoding mRNA allow the **comparison** between the two used promoters and the quantification of **RNA levels** by measuring a calibration curve. In addition, we **compare the growth curves** of the two cell cultures and perform **live/dead staining assays** to investigate the impact of Pveg controlling *rpsB* expression. This allows us to determine whether the **constitutive promoter** used, has an **influence on the growth or viability of the cells**. If the intended Pveg **promoter has a negative influence**, two solutions are possible:

The first possibility is to use an **inducible promoter system** of which the optimal inducer concentrations needs to be determined by titrating the inducer concentration[[24]](#cite_note-24). The second possibility is to use a **weaker or stronger constitutive promoter** from a **library** or **various ribosome binding site strengths**[[25]](#cite_note-25). In both cases, the same experimental setup as used for the investigation on Pveg is feasible to verify success of measures taken.

Our kill switch design contains a **second promoter, the quorum sensing promoter PdegQ**, which may also exhibit a **different expression strength** than the original *rspB*-controlling promoter. In the case of **PdegQ** the expression levels is depend on the **cell density** and thus on the growth phase. This complicates the previously described assay to investigate Pveg impact on *rspB* expression. Nevertheless, the same assay can be used but has to be carefully planned with particular attention on various cell density conditions in the samples assayed.

With the **MATLAB Simbiology** toolbox, we created an *in silico* [model of our kill switch](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling). The model relies on ordinary differential equations, which helped us to understand the kill switch even further.

## References

[[1] Albino A. Dias et al., An Easy Method for Screening and Detection of Laccase Activity, The Open Biotechnology Journal, 2017, 11 89-93, doi: 10.2174/1874070701711010089](https://www.researchgate.net/publication/319960611_An_Easy_Method_for_Screening_and_Detection_of_Laccase_Activity)

[[2] Meng-Hsuan Wu et al., Enhancement of laccase activity by pre-incubation with organic solvents, Nature Scientific Reports, 2019, 9:9754, doi: 10.1038/s41598-019-45118-x](https://www.nature.com/articles/s41598-019-45118-x)

[[3] iGEM TU Munich 2013: Erythromycin esterase (EreB)](https://2013.igem.org/Team%3ATU-Munich/Results/Recombinant%20)

[[4] J. Hudzicki, Kirby-Bauer Disk Diffusion Susceptibility Test Protocol, American Society for Microbiology, 2009](https://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf)
h
[[5] Merck: Laccase from  *Trametes versicolor*](https://www.sigmaaldrich.com/catalog/product/sigma/38429?lang=de&region=DE)

[[6] Morar, M., Pengelly, K., Koteva, K., & Wright, G. D. (2012). Mechanism and diversity of the erythromycin esterase family of enzymes. Biochemistry, 51(8), 1740-1751. Supporting information available via: https://pubs.acs.org/doi/10.1021/bi201790u [19.10.2020]](https://pubs.acs.org/doi/10.1021/bi201790u)

[[7] Huang, J., Liu, S., Zhang, C. et al. Programmable and printable *Bacillus subtilis* biofilms as engineered living materials. Nat Chem Biol 15, 34–41 (2019)](https://doi.org/10.1038/s41589-018-0169-2%E2%80%AF)

[[8] GeneGuard: a Modular Plasmid System Designed for Biosafety. Wright O, Delmans M, Stan GB, Ellis T. ACS Synth Biol. 2014 May 13. 10.1021/sb500234s](https://pubmed.ncbi.nlm.nih.gov/24847673/)

[[9] Gerwig J, Kiley TB, Gunka K, Stanley-Wall N, Stülke J. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in *Bacillus subtilis*. Microbiology (Reading). 2014 Apr;160(Pt 4):682-691.](https://europepmc.org/article/MED/24493247)

[[10] Diethmaier, Christine; Pietack, Nico; Gunka, Katrin; Wrede, Christoph; Lehnik-Habrink, Martin; Herzberg, Christina et al. (2011): A novel factor controlling bistability in *Bacillus subtilis*: the YmdB protein affects flagellin expression and biofilm formation. In: Journal of Bacteriology 193 (21), S. 5997–6007.](https://jb.asm.org/content/193/21/5997)

[[11] Alexander Gräwe, Jan Ranglack, Anastasia Weyrich, Viktor Stein, iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains, Nucleic Acids Research, Volume 48, Issue 4, 28 February 2020, Page e24](https://pubmed.ncbi.nlm.nih.gov/31925441/)

[[12] M.Y. Pavlov, R.E. Watts, Z. Tan, V.W. Cornish, M. Ehrenberg, A.C. Forster, Slow peptide bond formation by proline and other N-alkylamino acids in translation., Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 50–54.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629218/)

[[13] Radeck, J., Meyer, D., Lautenschläger, N. et al. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for *Bacillus subtilis*. Sci Rep 7, 14134 (2017).](https://doi.org/10.1038/s41598-017-14329-5)

[[14] Registry of Standard Biological Parts:Cre recombinase; Part:BBa\_K1680007, accessed on October 25rd 2020](http://parts.igem.org/Part%3ABBa_K1680007)

[[15] Registry of Standard Biological Parts: lox71 ; Part:BBa\_I718017, accessed on October 25rd 2020](http://parts.igem.org/Part%3ABBa_I718017)

[[16] Guiziou S., Sauveplane V., Chang H.J.,*et al*. A part toolbox to tune genetic expression in *Bacillus subtilis*. Nucleic Acids Research, 2016, 44, 7495–7508, https://doi.org/10.1093/nar/gkw624](https://academic.oup.com/nar/article/44/15/7495/2457776)

 [[17] Corish P., Tyler-Smith C., DNA sequencing at 40: past, present and future, Protein Engineering, Design and Selection,1999, 12, 1035–1040, https://doi.org/10.1093/protein/12.12.1035
 [[18] Shendure J., Balasubramanian S., Church G.M., *et.al.* , DNA sequencing at 40: past, present and future, Nature, 2017, 550: 345–353, DOI: 10.1038/nature24286](https://www.nature.com/articles/nature24286%20)

[[19] Hsiu-Man Lien, Hui-Yu Wu, Chiu-Lien Hung, et. al, Antibacterial activity of ovatodiolide isolated from Anisomeles indica against Helicobacter pylori, Scientific Reports, 2019, doi: 10.1038/s41598-019-40735-y](https://www.researchgate.net/publication/331680605_Antibacterial_activity_of_ovatodiolide_isolated_from_Anisomeles_indica_against_Helicobacter_pylori)

[[20] Shinichiro Shoji, Corey M. Dambacher, Zahra Shajani, et. al, Systematic Chromosomal Deletion of BacterialRibosomal Protein Genes, Journal of Molecular Biology, 2011, 413: 751-761, doi: https://doi.org/10.1016/j.jmb.2011.09.004](https://www.sciencedirect.com/science/article/abs/pii/S0022283611009934)

[[21] https://www.britannica.com/science/erythromycin, accessed on October 25rd 2020](https://www.britannica.com/science/erythromycin%20)

[[22] https://www.thermofisher.com/order/catalog/product/L7007#/L7007, accessed on October 25rd 2020](https://www.thermofisher.com/order/catalog/product/L7007#/L7007)

 [[23] Livak K.J., Schmittgen T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods, 2001, 25, 402-408, https://doi.org/10.1006/meth.2001.1262](https://www.sciencedirect.com/science/article/pii/S1046202301912629?via%3Dihub)

 [[24] Meyer A.J., Segall-Shapiro T.H., Glassey E. *et.al.*  Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors, Nature Chemical Biology, 2018, 15, 196–204, DOI: 10.1038/s41589-018-0168-3](https://www.nature.com/articles/s41589-018-0168-3%20)

[[25] Registry of Standard Biological Parts: Promoters/Catalog/B. subtilis/Constitutive, accessed on October 25rd 2020](http://parts.igem.org/Promoters/Catalog/B._subtilis/Constitutive)](https://academic.oup.com/peds/article/12/12/1035/1567742)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TU Darmstadt/Experiments

image/svg+xml

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O

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[![logo](https://static.igem.org/mediawiki/2020/4/47/T--TU_Darmstadt--black_owl.png)](https://2020.igem.org/Team%3ATU_Darmstadt)

* PROJECT

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Description)[BACKGROUND](https://2020.igem.org/Team%3ATU_Darmstadt/Background)
  [IMPLEMENTATION](https://2020.igem.org/Team%3ATU_Darmstadt/Implementation)[PHARMACEUTICAL DEGRADATION](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Pharmaceutical_Degradation)
  [BIOFILM](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Biofilm)
  [KILL SWITCH](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Kill_Switch)[NOTEBOOK](https://2020.igem.org/Team%3ATU_Darmstadt/Notebook)
  [EXPERIMENTS](https://2020.igem.org/Team%3ATU_Darmstadt/Experiments)
  [BACKBONES](https://2020.igem.org/Team%3ATU_Darmstadt/Project/Backbones)
* PEOPLE

  [TEAM](https://2020.igem.org/Team%3ATU_Darmstadt/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Attributions)
  [COLLABORATIONS](https://2020.igem.org/Team%3ATU_Darmstadt/Collaborations)
  [SPONSORING](https://2020.igem.org/Team%3ATU_Darmstadt/People/Sponsoring)
* MODELING

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Model)[ENZYME MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Enzyme_Modeling)
  [BIOFILM MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Biofilm_Modeling)
  [KILL SWITCH MODELING](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Kill_Switch_Modeling)[ROSETTA GUIDE](https://2020.igem.org/Team%3ATU_Darmstadt/Model/Rosetta_Guide)
* HUMAN PRACTICES

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Human_Practices)[INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3ATU_Darmstadt/Integrated_Human_Practices)
  [SCIENCE COMMUNICATION & EDUCATION](https://2020.igem.org/Team%3ATU_Darmstadt/Education)
  [RESPONSIBLE RESEARCH](https://2020.igem.org/Team%3ATU_Darmstadt/HP/Ethics)
* PARTNERSHIP

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership)[SUMMARY & METHODS](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Summary_And_Methods)
  [EXPERTS & LITERATURE](https://2020.igem.org/Team%3ATU_Darmstadt/Partnership/Experts_And_Literature)
* SAFETY

  [OVERVIEW](https://2020.igem.org/Team%3ATU_Darmstadt/Safety)[HOW TO HANDLE B-TOX](https://2020.igem.org/Team%3ATU_Darmstadt/Safety/How_To_Handle_B-Tox)
* ACHIEVEMENTS

  [AWARDS](https://2020.igem.org/Team%3ATU_Darmstadt/Awards)[BEST SOFTWARE](https://2020.igem.org/Team%3ATU_Darmstadt/Software)
  [BEST SUSTAINABLE DEVELOPMENT IMPACT](https://2020.igem.org/Team%3ATU_Darmstadt/Sustainable)[PARTS](https://2020.igem.org/Team%3ATU_Darmstadt/Parts)
  [CONTRIBUTION](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution)
  [ENGINEERING SUCCESS](https://2020.igem.org/Team%3ATU_Darmstadt/Engineering)
* JUDGING FORM

  [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3429)[POSTER](https://2020.igem.org/Team%3ATU_Darmstadt/Poster)

# Experiments

## Laboratory methods

Please take a look at our “Methods” on the right side. By clicking on the book, you can explore all our planned methods.

Unfortunately, due to the COVID-19 pandemic we were not able to actively work in our laboratory so most of these protocols were not used. However, most of the protocols were acquired from previous iGEM teams over the last years who developed them carefully. Therefore, we trust in their experience and we are sure that these would definitely work.

These “Methods“ will help you fully comprehend which experiments were planned to be done in the lab in detail. If you want to know more about our flow chamber you can find more details [here](https://2020.igem.org/Team%3ATU_Darmstadt/Contribution#Flow%20Chamber). In case you want to retrace our complete journey week by week, please do not hesitate to take a look at our [notebook](https://2020.igem.org/Team%3ATU_Darmstadt/Notebook).

[![figure](https://static.igem.org/mediawiki/2020/d/d1/T--TU_Darmstadt--Methods.png)

Click me](https://static.igem.org/mediawiki/2020/1/1f/T--TU_Darmstadt--Methods_TU_Darmstadt_2020.pdf)

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# Team:TU Darmstadt/Results