Team:TU Darmstadt/Poster

Poster: TU_Darmstadt

B-TOX: reduction of wastewater toxicity using a B. subtilis biofilm

Presented by Team TU_Darmstadt 2020

Authors: Philipp Becker¹, Jan-Philip 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.

Introduction

Micropollutants, like pharmaceuticals or chemical residues, in wastewater are a global issue. These substances pose an alarming and detrimental threat to the environment and can even lead to the extinction of species. For example, Diclofenac can cause liver and kidney damage in fish at low concentrations of 50 ng/L and therefore threat whole fish populations^[1]. In the end, even lives of people, animals and plants are severely affected and harmed^[2]. Even after intensive and costly purification in wastewater treatment plants, these pollutants are still present in purified water and have so far been unavoidably released and pollute our environment^[3,4]! We as humans have produced these detrimental substances. It is our responsibility to prevent their further release into the environment^[5].

This inspired us to create B-TOX!

B-TOX is an engineered modular Bacillus subtilis biofilm, with enzymes displayed on the extracellular matrix. These enzymes can neutralize many different micropollutants. Our solution utilizes laccase enzymes, more specifically CueO and CotA, which we immobilize in the extracellular matrix by fusing them to the major biofilm matrix component TasA. Diclofenac is our initial target as it should be transformed into a less toxic substance by CueO and CotA^[6]. Besides from CueO and CotA we utilize the esterase EreB to reduce azithromycin in wastewater^[7]. Therefore, B-TOX will reduce wastewater toxicity and is a contribution to the fight against global environmental pollution for a sustainable, clean and safe world.

References

[1] German Environment Agency: Arzneimittelwirkstoffe, https://www.umweltbundesamt.de/themen/wasser/fluesse/zustand/arzneimittelwirkstoffe#diclofenac, accessed on July 15th 2020
[2] German Environment Agency: Chemikalienwirkung, https://www.umweltbundesamt.de/daten/chemikalien/chemikalienwirkungen#prufen-der-umweltwirkung-von-chemikalien, accessed on July 15th 2020
[3] Frankfurter Allgemeine Zeitung: Medikamentenreste im Abwasser, https://www.faz.net/aktuell/wissen/medizin-ernaehrung/neue-klaertechniken-gegen-medikamentenreste-im-abwasser-16588998.html, accessed on November 8th 2020
[4] German Environment Agency: Schmerzmittel belasten deutsche Gewässer, https://www.umweltbundesamt.de/presse/pressemitteilungen/schmerzmittel-belasten-deutsche-gewaesser, accessed on July 15th 2020
[5] Hillenbrand et al., Maßnahmen zur Verminderung des Eintrages von Mikroschadstoffen in die Gewässer – Phase 2, Umweltforschungsplan des Bundesministeriums für Umwelt, Naturschutz, Bau und Reaktorsicherheit, 2016, Forschungskennzahl 3712 21 225
[6] Yu, H., Nie, E., Xu, J., Yan, S., Cooper, W. J., & Song, W. (2013). Degradation of diclofenac by advanced oxidation and reduction processes: kinetic studies, degradation pathways and toxicity assessments. Water research, 47(5), 1909-1918.
[7] 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.

Pharmaceutical Degradation

We utilize enzymes that can transform such substances into less toxic ones. We chose the two laccases CueO (E. coli) and CotA (B. subtilis) for degradation of the analgetic diclofenac, as well as the esterase EreB (E. coli) for degradation of antibiotics like erythromycin and azithromycin (see Fig. 1)^[1,2,3]. Laccases can oxidize phenolic compounds like in diclofenac, leading to less toxic substances. EreB is capable of breaking down erythromycin effectively^[4]. As EreB also displays some promiscuity towards azithromycin, we aim to utilized site-directed mutagenesis to increase the enzymes activity towards this specific substance^[5].

Fig. 1: Enzymatic transformation of micropolutants.

References

[1] Zeng J, Zhu Q, Wu Y, Lin X. Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere. 2016 Apr;148:1-7. doi: 10.1016/j.chemosphere.2016.01.019. Epub 2016 Jan 16. PMID: 26784443.
[2] Ma X, Liu L, Li Q, Liu Y, Yi L, Ma L, Zhai C. High-level expression of a bacterial laccase, CueO from Escherichia coli K12 in Pichia pastoris GS115 and its application on the decolorization of synthetic dyes. Enzyme Microb Technol. 2017 Aug;103:34-41. doi: 10.1016/j.enzmictec.2017.04.004. Epub 2017 Apr 21. PMID: 28554383.
[3] http://parts.igem.org/wiki/index.php?title=Part:BBa_K1159000
[4] https://2013.igem.org/Team:TU-Munich/Results/Recombinant
[5] Morar M, Pengelly K, Koteva K, Wright GD. Mechanism and diversity of the erythromycin esterase family of enzymes. Biochemistry. 2012 Feb 28;51(8):1740-51. doi: 10.1021/bi201790u. Epub 2012 Feb 10. PMID: 22303981.

Biofilm

We use a B. subtilis Biofilm to clean the wastewater by creating a fusion protein of the major biofilm matrix component TasA and enzymes such as CotA, CueO or EreB^[1,2]. When the function of both of these protein components is preserved, their fusion enables the biofilm to produce immobilized enzymes in high numbers all over the biofilm structure.

Another one of our efforts includes the deletion of the sigF gene encoding the sporulation sigma factor σ^F to completely disable any kind of sporulation which would decrease the structural integrity of the biofilm and the efficiency of enzyme production^[3]. Additionally, we want to create a knockout of the sinR gene to increase biofilm robustness.

References

[1] Branda, S.; Chu, F.; Kearns, D. (2006): A major protein component of the Bacillus subtilis biofilm matrix. In: Molecular microbiology 59 (4), S. 1229–1238, DOI 10.1111/j.1365-2958.2005.05020.x.
[2] 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), DOI 10.1038/s41589-018-0169-2.
[3] Kolter et al. (2013) Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol. 11(3): 157-168

Kill Switch

Our kill switch confines the organism by inhibiting the expression of the essential ribosomal gene rpsB hindering translation^[1]. We will replace the native promoter of rpsB with the degQ promoter (PdegQ)^[2], which is a quorum sensing (QS) controlled promoter.

QS signaling molecules are produced when cell density increases^[3]. At high cell density, the corresponding QS promoter PdegQ is active. When individual B. subtilis  cells leave the biofilm, cell density decreases and PdegQ is inactive, leading to lower expression levels of rpsB and thus resulting in cell death.

Because cell death in the growth phase is not desirable, we opted for the constitutive promoter Pveg in early stages of growth. When the right cell density is reached, the constitutive promoter is exchanged via Cre recombinase induced inversion of a combined promoter region (see Fig. 1)^[4].

Figure 1: Our kill switch cassette.

Further improvement of the kill switch cassette would be achieved through the use of an inducible promoter (PcymRC) for the growth phase^[5]. After the promoters are inverted, cells are transferred into a fresh medium. The inducer is no longer added and the kill switch is active.

Figure 2: Our improved kill switch cassette.

In addition, we will introduce GFP coding sequence on the upstream antisense strand (See Fig. 2) for verification of a functional kill switch.

References

[1] Geisser, M., Tischendorf, G.W. & Stöffler, G. Comparative immunological and electrophoretic studies on ribosomal proteins of Bacillaceae. Molec. Gen. Genet. 127, 129–145 (1973).
[2] Bingyao Zhu, Jörg Stülke, SubtiWiki in 2018: from genes and proteins to functional network annotation of the model organism Bacillus subtilis, Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018
[3] Matthew R. Parsek, E.P. Greenberg, Sociomicrobiology: the connections between quorum sensing and biofilms, cell.com, 2005, Volume 13 issue 1, P27-33
[4] Zhang, Zuwen, and Beat Lutz. “Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein.” Nucleic acids research vol. 30,17 (2002): e90. doi:10.1093/nar/gnf089
[5] Meyer, A.J., Segall-Shapiro, T.H., Glassey, E. et al. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat Chem Biol 15, 196–204 (2019).

Modeling

CotA Modeling

We used the Rosetta docking protocol to find a plausible conformation of the enzyme substrate complex^[1].

The images show the conformation of the enzyme substrate complex with the best interface score. The model indicates there are two threonine interacting strongly with diclofenac.

To get a deeper insight in diclofenac docking we ran a docking simulation with the laccase from T. vesicolor, an enzyme reported to transform diclofenac^[1]. Based on both runs we conclude that the CotA laccase could be improved by adding aspartate and phenylalanine residues in similar positions like the T. vesicolor laccase.

EreB Modeling

Protein function is deeply related to protein structure. Since there was no crystal structure of EreB, we run a RosettaCM simulation to identify a plausible structure.

Fusion Proteins modeling

In a second step we repeated the procedure to get a possible structure of the TasA-CotA-fusion protein and the EreB-TasA-fusion protein.

Both structures seem to be stable.

Killswitch Modeling

We modeled the kill switch reaction pathways. The following assumtions have been made:

1. The pathways of the quorum-sensing system can be described as rate of changes.
2. The model is built around mass-action equations.
3. One cell stands for an undefined cluster of cells.
4. The extracellular ComX concentration needs to be above a certain threshold to be used by ComP to phosphorylate ComA.
5. The educts for pre-ComX and ComA are produced continuously.

Biofilm modeling

We came together with iGEM Hannover to build a software able to simulate biofilm growth. We used physical laws to model bacteria growth and interactions and probability theory to model biological phenomena. Comparing our results with literature values we concluded that the results seem to be close to real world data^[2].

References

[1]Leticia Arregui et al., Laccases: structure, function, and potential application in water bioremediation, Microbial Cell Factories, 2019, 18:200, doi: 10.1186/s12934-019-1248-0
[2]van Heerden, J.H., Kempe, H., Doerr, A. et al. Statistics and simulation of growth of single bacterial cells: illustrations with B. subtilis and E. coli . Sci Rep 7, 16094 (2017). https://doi.org/10.1038/s41598-017-15895-4

Methods

ABTS Assay

After enzyme production in E. coli, we plan to prove the activity of the laccases in vitro. We planned on using an Assay with 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 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^[1].

Kirby-Bauer Assay

Measurement of activity was also planned for EreB by performing a Kirby-Bauer-Assay as done from iGEM TU Munich 2013. It is a disk diffusion test, where the ability of bacteria to grow on plates containing the antibiotic erythromycin or azithromycin with and without previous treatment of EreB is being measured^[2].

Toxicity Assay

As proof that our degraded substances are noticeably less toxic for aquatic organisms, we planned on performing a Zebrafish Embryo Toxicity Assay^[3].

Flow Chamber

We designed a flow chamber for comparing different bacterial strains regarding their biofilm stability. Our flow chamber is specifically designed to direct a liquid (e.g., phosphate-buffered saline) to flow over a biofilm in a thin layer, simulating the most extreme conditions in a wastewater treatment plant. We also developed a microplate reader assay for subsequent analysis. Our assay allows us to estimate the ratio of cells that have been washed out compared to all cells in the biofilm.

References

[1] Albino A. Dias, António J.S. Matos, Irene Fraga, Ana Sampaio, Rui M.F. Bezerra (2017) An Easy Method for Screening and Detection of Laccase Activity, The Open Biotechnology Journal, 10.2174/1874070701711010089
[2] https://2013.igem.org/Team:TU-Munich
[3] Schmidt S., Busch W., Altenburger R., Küster E. (2016). Mixture toxicity of water contaminants-effect analysis using the zebrafish enbryo assay (Danio reria), Chemosphere, Volume 152, June 2016, Pages 503-512

Human Practices

To round off our project, it was essential to address the public – including stakeholders, and various experts in life science. With the focus on doing responsible research and improving our project, we thought through every step of our project and got in contact with various experts. To summarize our work in the field of Integrated Human Practices we clustered all the experts and the information we gathered into the following four categories:

• Environment: Getting in contact with the German federal environment agency (UBA) as well as an ecotoxicologist helped us to understand the problem of micropollutants in wastewater and how difficult it is to detect the exact effects on an ecosystem.
• Synthetic Biology: The information provided by experts regarding laccases or B. subtilis helped us to elaborate our project in detail theoretically and extend our knowledge on these topics.
• Implementation: Visits of wastewater treatment plant (WWTPs) and interviews with stakeholders as well as a professor already working on using biofilms in WWTPs allowed us to plan and calculate the implementation of our “B-TOX”.
• Ethics: We discussed with professors of philosophy and a member of the ethic commission of our university about our project, risk evaluation, ethical aspects and how to make sure that our research is responsible and ethically justifiable.

To reach out to the society we did a survey asking about knowledge concerning synthetic biology (SynBio) and for example the acceptance of genetically modified organisms (GMOs) in WWTPs. Only about half of the participants knew what SynBio is, leading us to further focus on science communication.

We created a podcast called “Genomenal” where we explain different topics of biotechnology in an easy and understandable way. Moreover, we are talking about our project and the iGEM competition.

For younger parts of the society we created the minigame “The Genomenal Adventures of Dr. W”, where each level represents and explains a basic laboratory method.

Implemetation

We want to implement our B. subtilis biofilm in wastewater treatment plants (WWTPs) by adding another purification stage subsequent to the final clarifier. Our solution uses biofilm carriers on which we grow our engineered B. subtilis biofilm. These are small floating bodies with structures that on the one hand provide large surface for biofilm attachment and on the other hand protection against mechanical forces.

Since we are using a genetically engineered B. subtilis strain and plan to implement it into a WWTP we have to deal with biosafety and biocontainment issues. In order to minimize risks to humans and to the environment, we have taken the following precautions:

1) Introducing a kill switch

We developed a kill switch to ensure our bacteria will not be able to survive outside the biofilm.

2) Engineering of B. subtilis strain

We knocked out two genes two icrease the biofilm stability.

3) Safety form for the use of "B-TOX"

We want to make sure that our project is handled responsibly and safe. Therefore, we have developed a safety form that has to be filled out if people want to use “B-TOX”.

4) How to handle “B-TOX”

Using GMOs in WWTPs is a new approach which is not in application yet. Employees who are going to work with “B-TOX” may have never worked with GMOs before. We therefore made a manual with the description of risks, a safety protocol for employees and the step by step practical application.

Contributions

In order to enable the iGEM community to benefit from our work and our experience, we have carefully documented the same and written instructions.

Future teams can…

• build their own flow chamber using a 3D-Printer to simulate water flow with our blueprint.
• easily start their own podcast using our instructions.
• do simulations with our guide on how to use Rosetta.
• use our project as we provided an instruction, written in an easy understandable way to make it accessible for a wide audience.


• In partnership with Kaiserslautern and Stuttgart, called the Oxiteers, we provide information pages on our projects, experts and literature to make it easy for future iGEM teams to build upon our work.
• Moreover, we contributed information and modeling data to several existing parts (EreB: BBa_K1159000, Cre recombinase: BBa_K1680007) and created 10 basic parts and one composite part (TasA-EreB fusion protein: BBa_K3429013).

Achievements & Awards

Achievements:
We achieved all bronze and silver criteria and for gold we fulfilled 4 out of 7 criteria as:

• Integrated Human Practices
• Project Modeling
• Partnership
• Science Communication

Awards:

Best Education
We broadcasted a livestream with iGEM Kaiserslautern, published an article in an German scientific journal, created a podcast and a minigame (together with Aleksa Zečević) to educate about synbio in an easy and understandable way. With all this work, we wanted to make biotechnology more tangible and accesible, thus providing ways to communicate society’s opinion to us via survey or e-mail.

Best Integrated Human Practices
We reached out to several experts, stakeholders and the society and integrated their input in our project. For example, we build a kill switch to make our project safe and have it accepted by society. To prevent misuse of our project we created a safety form and a guide on how to use our project.

Best Model
We developed a software to model biofilm growth in collaboration with iGEM Hannover. Additionally, we modeled our quorum sensing-based kill switch mechanism, determined possible 3D structures for EreB and validated the stability of our enzyme structure, predicted the structure of our fusion proteins of TasA and one of our degradation enzymes and simulated their binding affinity to their targets.

Best Software
We developed a software tool in collaboration with the iGEM Team Hannover which enables the user to run a molecular dynamics simulation of biofilm growth. It includes a three class and utility function. The code is open for everyone to use and our Wiki provides a detailed guide.

Best Sustainable Development Impact
The focus of our project to reduce wastewater pollution by pharmaceuticals is in strong agreement with the United Nation’s sustainable development goals 6 and 14. With our adaptable, easy to use and cost efficient project we contribute to ensure access to water and sanitation for all (6) and to prevent harm to our marine ecosystems (14).

Outlook

But our project does not end here. There are still experiments to make and possibilities to be explored. Here is a little outlook into to future:

Possible future wet-lab experiments include:

• Purification and in vitro testing of our enzymes (wildtype and improved variants)
• Expression of our enzymes as TasA fusion protein within a B. subtilis biofilm
• Testing our biofilm enzymes via an ABTS-assay
• Testing the stability of our biofilm with ATM and flow chamber experiments

Other applications:
Our biofilm could also be used in different applications. Through changing the Biofilm-Carrier or the enzymes fused to TasA, new apllications emerge. For example it could be used to fight dye pollution, that are produced through textile factories in some countries. Another possibility would be to expresse a fusion protein with PETase or MHETase to fight micro plastic polution.
Our biofim could even be used in space someday. We talked with the “Deutsches Zentrum für Luft- und Raumfahrt” (DLR) and they told us that experiments for biofilm groth in space are already planed. Maybe someday biofilms will be used on spaceships- and stations.

Acknowledgements

Here you can see all the great people who helped us during this year:

Primary PI: Professor Dr. Heribert Warzecha​
Secondary PI: Professor Dr. Johannes Kabisch​
Our advisors: Fran Bacic Toplek,​ Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, Leon Kraus, Chris Sürder and Maximilian Zander​

Others:
• Dr. Christian Dietz​
• Angelina Folberth
• PD Dr. Stefan Immel​
• Jörg Kalkowski​​
• PD Dr. Arnulf Kletzin​
• Dr. Melanie Mikosch-Wersching​
• Prof. Dr. Boris Schmidt​
• Prof. Dr. Viktor Stein
• Prof. Dr. Torsten Waldminghaus​​
• Barbara Wolf​​
• Prof. Dr. Nico 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. Dr. Andreas Jürgens​
• Prof. Dr. Susanne Lackner​​
• Prof. Dr. Ralf Möller​
• Prof. Dr. Alfred Nordmann​
• Prof. Dr. Jörg Oehlmann
• Dr. Sabine Sané
• Dr. Dietmar Schlosser
• Dr. Patrick Schröder
• Thomas Seeger​
• Prof. Dr. Jörg Stülke

Sponsors