Presented by Team TU_Darmstadt 2020
Authors: Philipp Becker¹, Jan-Philipp Kahl¹, Jan Kalkowski¹, Robert Klein¹, Rosi Krebs¹, Angela Kühn¹, Mehryad Mataei¹, Johanna Möller¹, Jonas Müller¹ and Max Schäfer¹
¹iGEM Student Team Member, Departement of Biology, Technical University (TU) of Darmstadt, Darmstadt, Germany
Project Abstract:
Water is undoubtedly one of our most precious goods and basis of life. But somehow, we humans have managed to neglect and pollute this meaningful resource. Nonetheless, most of us aren’t even aware of the consequences.
In this year's iGEM project, we have made it our mission to make a difference in wastewater treatment and develop an innovative future for pharmaceutical degradation: B-TOX, a modular biofilm able to degrade a variety of detrimental micropollutants like the anti-inflammatory drug diclofenac.
By devising an enhanced and modular B. subtilis biofilm, we can render pharmaceutical residues less toxic, utilizing the degrading properties of enzymes. We immobilize our degradation enzymes in the extracellular biofilm matrix, thereby providing a self-sustaining system without the necessity downstream processing.
A safe implementation and the prevention of bacteria release is given through our kill switch system, connecting the survival of our bacteria to the presence of determined molecules.
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<div class="title">B-TOX: reduction of wastewater toxicity using a <i>B. subtilis</i> biofilm </div> | <div class="title">B-TOX: reduction of wastewater toxicity using a <i>B. subtilis</i> biofilm </div> | ||
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| − | <div class="text"> | + | <div class="text">Presented by Team TU_Darmstadt 2020 <br><br> |
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| + | Authors: Philipp Becker¹, Jan-Philipp Kahl¹, Jan Kalkowski¹, Robert Klein¹, Rosi Krebs¹, Angela Kühn¹, Mehryad Mataei¹, Johanna Möller¹, Jonas Müller¹ and Max Schäfer¹<br><br> | ||
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Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, | Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, | ||
Leon Kraus, Chris Sürder and Maximilian Zander | Leon Kraus, Chris Sürder and Maximilian Zander | ||
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<li> Angelina Folberth, </li> | <li> Angelina Folberth, </li> | ||
<li> Dr. Immel, </li> | <li> Dr. Immel, </li> | ||
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<li> Jörg Kalkowski, </li> | <li> Jörg Kalkowski, </li> | ||
<li> PD Dr. Kletzin, </li> | <li> PD Dr. Kletzin, </li> | ||
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<li> Dr. Mikosch-Wersching, </li> | <li> Dr. Mikosch-Wersching, </li> | ||
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<li> Prof. Schmidt, </li> | <li> Prof. Schmidt, </li> | ||
<li> Prof. Stein, </li> | <li> Prof. Stein, </li> | ||
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<li> Florian Heyn, </li> | <li> Florian Heyn, </li> | ||
<li> Wolfgang John, </li> | <li> Wolfgang John, </li> | ||
| + | <li> Prof. Jürgens, </li> | ||
| + | <li> Prof. Lackner, </li> | ||
<li> Prof. Dr. Ralf Möller, </li> | <li> Prof. Dr. Ralf Möller, </li> | ||
| + | <li> Prof. Nordmann, </li> | ||
<li> Prof. Dr. Jörg Oehlmann, </li> | <li> Prof. Dr. Jörg Oehlmann, </li> | ||
<li> Dr. Sabine Sané, </li> | <li> Dr. Sabine Sané, </li> | ||
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Revision as of 12:52, 8 November 2020
Poster: TU_Darmstadt
B-TOX: reduction of wastewater toxicity using a B. subtilis biofilm
Introduction
Introduce your project and your team's goals.
Pharmies
We were concentrating on the problematic substances diclofenac (analgetic) and azithromycin (antibiotic). Both substances pose environmental hazards in high concentrations. They reach our wastewater in large quantities. At wastewater treatment plants, only a small part can be degraded endangering aquatic fauna and flora. Other substances, such as ibuprofen or estrogen are also problematic.
We utilize enzymes that can modify such substances rendering them less toxic. We chose the two laccases CueO (E. coli ) and CotA ( B. Subtilis ) for diclofenac, as well as the esterase EreB ( E. coli ) for antibiotics like erythromycin and azithromycin. Laccases are oxidoreductases that can oxidize phenolic compounds like in diclofenac, as well as other pharmaceuticals, such as carbamazepine, 17β-Estradiol. The resulting products are demonstrably less toxic. EreB has already been shown to being capable of breaking down erythromycin effectively. EreB also displays some promiscuity towards azithromycin. To increase the activity of EreB towards azithromycin we want to utilize site-directed mutagenesis.
With the selected enzymes we are already able to degrade a wide range of pharmaceuticals. Besides the already mentioned substances, chloramphenicol, bisphenol A and 4-nonylphenol can also be oxidized by laccase.
Enabling us to adapt our biofilm to different local conditions of wastewater pollution. Because of this B-TOX is not restricted to one area.
Biofilm
Where is the biofilm? Why is the Biofilm? How is the Biofilm
Killswitch
How are you going to solve the problem? Where did the idea come from?
Modeling
Here will be modeling stuff somtimes in the future.
Methods
Measurement of Enzyme Activity
After enzyme production in E. coli, we plan to prove the activity of the laccase in vitro. We planned on using an Assay with 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS). ABTS is a widely used substrate for laccase activity assay. By enzymatic oxidation, it forms a stable radical cation, which can be measured photometrically at 420 nm and thus allows the measurement of laccase activity.
Measurement of Antibiotic Degradation
As done for the laccase, measurement of activity was also planned for EreB by performing a Kirby-Bauer-Assay as done from iGEM TU Munich 2013. The Kirby-Bauer-Assay is a disk diffusion test. While using model bacteria, the ability of these bacteria to grow on plates containing the antibiotics erythromycin or azithromycin with and without previous treatment of EreB is being measured.
Toxicity Assay
As proof that our degraded substances are noticeably less toxic for aquatic organisms, we planned on performing a Zebrafish Embryo Toxicity Assay. Zebrafish embryos are widely used model organisms for aquatic toxicity as they are not classified as animal testing (within the first 5 days after fertilization). This way, we are able to determine acute toxicity and teratogenicity.
Flow Chamber
Lorem ipsum
References
Human Practices
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Implemetation
some WWTP stuff i guess
Contributions
Use this section to explain whatever you would like! Suggestions: Just Contributions.
[O.K.]
[O.K.]
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Achievments & Awards
Use this section to explain whatever you would like! Suggestions:Achievments
Outlook
Use this section to explain whatever you would like! Suggestions: Outlook
Acknowledgements
Here you can see all the great people who helped us during this year.
Primary PI: Prof. Warzecha
Secondary PI: Prof. Kabisch
Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, Leon Kraus, Chris Sürder and Maximilian Zander
Primary PI: Prof. Warzecha
Secondary PI: Prof. Kabisch
Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, Leon Kraus, Chris Sürder and Maximilian Zander
- Others:
- Dr. Dietz,
- Angelina Folberth,
- Dr. Immel,
- Jörg Kalkowski,
- PD Dr. Kletzin,
- Dr. Mikosch-Wersching,
- Prof. Schmidt,
- Prof. Stein,
- Prof. Waldminghaus,
- Barbara Wolf,
- Prof. van der Vegt,
- Aleksa Zecevic,
- Working group of Prof. Kabisch and Prof. Warzecha,
- People from „Krautnah“ and from Hda of TU Darmstadt
- Former iGEMers
- Klara Eisenhauer,
- Jonathan Funk,
- Peter Gockel,
- Jamina Gerhardus,
- Robin Johannson,
- Thea Lotz,
- Tim Maier,
- Benjamin Meyer,
- Jean Victor Orth,
- Hannah Rainer,
- Lara Steinel,
- Leon Werner,
- Marius Wollrab
- Experts
- Dipl. Ing. Udo Bäuerle,
- PhD Yunrong Chai,
- Dr. Ulrich Ehlers,
- Prof. Dr. Sibylle Gaisser,
- Florian Heyn,
- Wolfgang John,
- Prof. Jürgens,
- Prof. Lackner,
- Prof. Dr. Ralf Möller,
- Prof. Nordmann,
- Prof. Dr. Jörg Oehlmann,
- Dr. Sabine Sané,
- Dr. Dietmar Schlosser,
- Dr. Patrick Schröder,
- Thomas Seeger,
- Prof. Dr. Jörg Stülke
Sponsors