Why did we talk to him?

• Patrick Schröder is a scientific administrator at the German Environment Agency. He is specialized in biodegradation of pharmaceuticals and the development and spread of antimicrobial resistances in the environment.

• He brought common mistakes in disposal of pharmaceuticals to our attention. In many regions in Germany it is sufficient for them to be disposed of via household waste, but in others they must be returned to designated collection facilities or pharmacies. In Germany, consumers can find out the correct disposal method on arzenitmittelentsorgung.de
• He also told us about pharmaceuticals that cause an immense burden on the environment due to their high consumption such as ibuprofen and hormones like estrogen. These pose risk to aquatic flora and fauna.
• Furthermore, he pointed out that diclofenac, a substance that we focus our attention on, has never undergone an environmental risk assessment as of today since it was authorized before 2005, like many other pharmaceuticals.

• In order to bring the information about proper disposal of pharmaceuticals to the broader public, we translated the flyer from the German Environment Agency into English, in order to increase the awareness of the general public internationally.
• We also decided to take a closer look at the degradation of estrogen and ibuprofen by laccases. Since laccases also are able to transform both substances, we try to make our contribution here.
• Our goal is to educate the citizens. If illness or symptoms exist, it is necessary to use pharmaceuticals in the process of recovery, but excessive consumption leads to environmental pollution and species extinction.[5]

text über Prof. Dr. Jörg Oehlmann

Why did we talk to him?

• Jörg Stülke is the Head of the Department of General Microbiology at the Institute of Microbiology and Genetics at the University of Göttingen. He is an expert for B. subtilis and designed a strain where the genes sinR and tasA are knocked out (GP1622).^[1] This strain would be useful for testing, if we could bring our enzymes as fusion proteins with TasA into the biofilm matrix. (Verlinkung Text TasA sinR knockout)

What information did we gather?

• Besides providing us the needed strain GP1622, he agreed with our assumption, that a knockout of sinR would not have any bad impacts on our biofilm.

How did we adjust our project?

• Getting the GP1622 B. subtilis strain enabled us to design our experiments and would have shortened the needed time in the lab.

Why did we talk to her?

• Dr. Sabine Sané did her subsequent PhD studies at the department of Microsystems Engineering at the University Freiburg and investigated biotechnological approaches to regenerative energy systems. Among other things, her research also concentrated on the degradation of micropollutants in wastewater. Therefore, her work and experience with laccases made her a great asset for our project. Sané was interviewed by our friends from iGEM Kaiserslautern.
  Sané mainly worked with the laccase of the fungus Trametes versicolor.

What information did we gather?

• The purification of laccases is very laborious and expensive, which is why we would benefit from avoiding it. An ABTS assay can be used to detect the most efficient laccase concentration.
• The degradation of estrogen via laccases is theoretically possible and was proved successful with the laccase of Trametes versicolor.


• The pH optimum of the laccases needs to be considered and the pH of the wastewater must eventually be adjusted accordingly.

How did we adjust our project?

• We were able to gather information in the conversation with Dr. Sané, thanks to the framework of our partnership with iGEM Kaiserslautern and iGEM Stuttgart (als Link). As a result, all her input has helped us to acquire more knowledge about the functionality of laccases, like the degradation of estrogen, which is also a problem in wastewater (Verlinkung liste pharmis). For our experimental approach (verlinkung zu experiments) we can resort to her contribution.

Why did we talk to him?

• PhD Yunrong Chai is an assistant professor at the College of Science at the Northeastern University in Boston and an expert for microbial genomics and biofilm formation. We talked to him about our ideas to engineer and improve our biofilm.

Which information did we gather?

• Concerning our plan to knockout the sinR gene of our B. subtilis to simplify the induction of the biofilm formation, PhD Chai explained that this knockout will make our biofilm more robust. The effects on other properties of our B. subtilis and our biofilm should be minor.
• Endospores are not able to produce our degradation enzymes, which is why preventing sporulation of our B. subtilis would be useful. As the sigma factor F (σ^F) regulates genes associated with sporulation, he suggested a sigF knockout to avoid sporulation. PhD Chai predicted no impact on gene expression outside of sporulation.
• The combination of a sigF and sinR knockout would lead to a B. subtilis that can not sporulate and forms a more robust biofilm.
• A fusion protein of the matrix protein TasA with our enzymes would be a perfect way to display the degradation enzymes in the biofilm matrix. Because TasA is very abundant, many enzymes would be located in the matrix. Furthermore, he successfully generated fusion proteins of TasA with fluorescence proteins in his lab.
• A mixed biofilm of different microorganisms could increase the robustness of our biofilm but would also complicate our assays and would be an advanced step in your project.

How did we adjust our project?

• We continued planning our fusion proteins with TasA and the knockout of sinR. Additionally, we integrated the knockout of sigF into our project to avoid sporulation of our B. subtilis.

Why did we talk to him?

• Dr. Dietmar Schlosser is group leader for Environmental Mycology in the Department of Environmental Microbiology at the Helmholtz Centre for Environmental Research in Leipzig. Although he is more of an expert for fungal laccases, we still considered his experience with laccases in general as valuable. All given answers and input from below were gathered from the interview protocol provided by iGEM Kaiserslautern, since they spoke directly with Dr. Schlosser and offered to ask our question in collaboration

Which information did we gather?

• Dr. Schlosser has confirmed the native function of laccases: their ability to detoxify. In fungal laccases that reaction leads to the formation of alkaline compounds.
• The laccase has a poor water solubility.
• The reaction time of an ABTS assay to determine the most efficient laccase concentration can range from a few days up to two weeks. As optimal concentration of ABTS he recommended 2 mM.
• To monitor the degradation of the starting materials, Dr. Schlosser uses the method of UHPLC coupled with MS/MS in his research.
• The public opinion on GMOs reveals a dread of unprecedented affects but an acceptance when used for medicinal applications.
• He investigates the toxicity of products by means of bacterial inhibition tests, which provide an EC₅₀ value.

How did we adjust our project?

• The information we gained from the conversation with Dietmar Schlosser within the framework of our partnership with iGEM Kaiserslautern and iGEM Stuttgart has helped us to develop further understanding for working with laccases. In our experimental planning we can resort to the bundled knowledge we have collected, as Dr. Schlosser’s approach on the tracing of product degradation is similar to our approach with HPLC. In correspondence to his toxicity assessments, we are confirmed in our experimental approach.

Why did we talk to her?

• Professor Dr. Lackner is the head of the department for sewage management at the TU Darmstadt since 2016. She is known for her work with microbial communities regarding wastewater purification in Germany, but also abroad, which is why an interview with her was of great interest for us. More so compelling was the fact that she has significant experience with the degradation of harmful pharmaceutical residues.

What information did we gather?

• Prof. Lackner told us about possible complications regarding GMOs in wastewater treatment plants and the difficulties surrounding this topic. She could confirm the danger pharmaceuticals pose in wastewater, among them diclofenac, as she has been working on its degradation herself.
• Hence, she was able to assure us that an oxidation of diclofenac was more than possible – as this has already been done. For this, it was even more helpful that she sent us continuative literature regarding diclofenac, as one of her Ph.D. students has worked on degrading the substance.
• When asked about other harmful substances, Prof. Lackner mentioned ibuprofen and carbamazepine as well as others.
• Due to her vast experience, she also gave us immense input regarding a carrier material for our biofilm and told us about the most efficient environmental conditions.

How did we adjust our project?

• She did not only confirm the alarming toxicity of diclofenac and that there is a need of a project idea like ours, but also pointed us in the direction of searching for other substances like carbamazepine.
• Her willingness to be at our disposal regarding literature or information about diclofenac and wastewater purification made her a great asset for our project. Thanks to her, and her research group, we were able to get crucial information on how and to which extent diclofenac can be oxidized by laccases.
• Since we were undecided on which carrier material we should apply for our biofilm, her recommendation of the ones she uses and the offer to provide us with them has meant a great deal for the thrive of our project.

• Already in the early phase of our project we knew that wastewater treatment plants would (WWTPs) be the main users of our project. Therefore, we had to find an expert who can introduce us into the purification stages and help us to find the best way to implement our biofilm. Udo Bäuerle studied construction engineering and focused on water management as well as on wastewater treatment. With his company B&P Beratende Ingenieure he constructed a lot of WWTPs.

Which information did we gather?

• He told us about the reoccurring problems associated with commonly used procedures like ozonolysis and the use of active carbon.
• Udo Bäuerle assured us that a project like ours would be really helpful and advised us to implement our system in an additional clarifier as fourth purification step. Our engineered Bacillus subtilis probably will not survive in the activated sludge clarifier which is the second purification step.
• Udo Bäuerle was the first person to mention rotating disc filters on which our biofilm could grow on and could be implemented into the WWTP.

How did we adjust our project?

• On this basis we were able to discuss our project with plant managers of our local WWTPs. We did research about the diving bodies and after discussing this idea with other experts we finally came to the biofilm carriers we want to use.