Revision as of 17:30, 9 November 2020

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^[4]. In the end, even lives of people, animals and plants are severely affected and harmed^[1]. 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^[2,^3]! 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: Chemikalienwirkung, https://www.umweltbundesamt.de/daten/chemikalien/chemikalienwirkungen#prufen-der-umweltwirkung-von-chemikalien, accessed on July 15th 2020
[2] 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
[3] German Environment Agency: Schmerzmittel belasten deutsche Gewässer, https://www.umweltbundesamt.de/presse/pressemitteilungen/schmerzmittel-belasten-deutsche-gewaesser, accessed on July 15th 2020
[4] German Environment Agency: Arzneimittelwirkstoffe, https://www.umweltbundesamt.de/themen/wasser/fluesse/zustand/arzneimittelwirkstoffe#diclofenac, 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 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

Kill Switch

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

QS signaling molecules are produced when cell density increases ^[4]. At high cell density, the corresponding QS promoter PdegQ is active. When individual B. subtilis cells leave the biofilm, cell density in their environment is lower and the PdegQ promoter becomes 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 the combined promoter region (see Fig. 1).

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 ^[9]. After the promoters are changed during Cre recombination, cells are transferred into a fresh medium. The inducer is no longer added, leading to cell death of any cells, that did not switch the promoter controlling the expression of the essential gene.

Figure 2: Our improved kill switch cassette.

In addition, we will add a GFP coding sequence on the upstream antisense strand (See Fig. 2) for easy 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]Iztok Dogsa, Kumari Sonal Choudhary, Ziva Marsetic et al, ComQXPA Quorum Sensing Systems May Not Be Unique to Bacillus subtilis: A Census in Prokaryotic Genomes, plos one, 2014, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096122
[4] Matthew R. Parsek, E.P. Greenberg, Sociomicrobiology: the connections between quorum sensing and biofilms, cell.com, 2005, Volume 13 issue 1, P27-33
[5] 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, http://www.subtiwiki.uni-goettingen.de/v3/gene/view/40C1E81BAB04BD1CC98FC57DF25D27DBDFEB5A59
[6] 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 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
[7] http://parts.igem.org/Part:BBa_K733002
[8] Silvia Fernández, Silvia Ayora, Juan C Alonso, Bacillus subtilis homologous recombination: genes and products, Research in Microbiology, 2000, 151,481-486 https://www.sciencedirect.com/science/article/pii/S0923250800001650?via%3Dihub
[9] 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.

The five best scoring structures

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. we assumed the following assumptions:

The pathways of the quorum-sensing system can be described as rate of changes.
The model is built around mass-action equations.
One cell stands for an undefined cluster of cells.
The extracellular ComX concentration needs to be above a certain threshold to be used by ComP to phosphorylate ComA.
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 phenomenon. In the following we present some plots from a simulation of B. subtilis.

Comparing our results with literature values we concluded that the results seem to be close to real world data.²

Methods

ABTS Assay

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.

Kirby-Bauer Assay

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

A critical issue for our project is the stability of the biofilm. For every new enzyme we want to display in the biofilm, we have to prove that the biofilm matrix component TasA is still functional and able to link Bacillus subtilis cells to provide a stable biofilm. In addition, we have to make sure that no cells escape into the environment when being exposed to the rough conditions that prevail in a WWTP. We therefore 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. We created a manual and contribute the files for 3D-printing to the iGEM community. Our approach is a low cost and highly modular system that can be attractive to other teams for various applications.

References

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.

ABBILDUNG biofilm carrier & abbildung Elisa
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

Additionally, we knock-out two genes in order to increase biosafety. The knock-out of the gene for transcription factor SinR will prevent cells from dispersing the biofilm. Knock-out of the sigF gene will provide a biofilm in which the cells are not able to sporulate.

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.

These safety aspects in mind, we had contact with the Federal Office for Consumer Protection and Food Safety of Germany (BVL) and were able to ask specific questions regarding the use of GMOs in WWTPs. Based on this and on the Annex III Part A of the Genetic Engineering Safety Ordinance – GenTSV we collected all the needed requirements and calculated the costs for “B-TOX” and came to the conclusion that our strategy is much more cost efficient than comparable methods.

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).

Achievments & 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, provide a podcast (together with Aleksa Zečević) and a minigame to educate about synbio in an easy understandable way. With all these work we provide open access to easy understandable information and also provided ways to communicate society’s opinion to us via survey or 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 save and more accepted from 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.

Outlook

But our project doesn't 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.

How can future iGEM Teams use our Project:
In the course of our project we collected a lot of Information about lacasses, B. subtilis and waste water treatment plants. We planed parts and experiments to test the ability of a B. subtilis biofilm to degrade substrates. This knowledge and in theorie designed parts and experiments can be used by future iGEM Teams to make their own biofilm projects and give it their own spin.

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

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 			<div class="text">
+Micropollutants, like pharmaceuticals or chemical residues, in wastewater are a global issue. These substances pose an <b>alarming and detrimental threat to the environment</b> 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<sup id="cite_ref-4"><a href="#cite_note-4">[4]</a></sup>. In the end, even <b>lives of people, animals and plants are severely affected and harmed</b><sup id="cite_ref-1"><a href="#cite_note-1">[1]</a></sup>. Even after intensive and costly purification in wastewater treatment plants, these pollutants are still present in purified water and have so far been <b>unavoidably released and pollute our environment</b><sup id="cite_ref-2"><a href="#cite_note-2">[2, </a></sup><sup id="cite_ref-3"><a href="#cite_note-3">3]</a></sup>! We as humans have produced these detrimental substances. It is our responsibility to prevent their further release into the environment<sup id="cite_ref-5"><a href="#cite_note-5">[5]</a></sup>.
+<br> <br>
-Micropollutants in wastewater are a global issue. These are anthropogenic substances like pharmaceuticals or chemical residues in general that pose an <b>alarming threat to the environment</b> and can even lead to the <b>extinction of species</b>. Various studies have been published that show concrete effects on the aquatic environment and therefore prove ecotoxicity among others <sup id="cite_ref-1"><a href="#cite_note-1">[1]</a></sup>. In total the <b>lives of people, animals and plants are severely affected and harmed</b> <sup id="cite_ref-1"><a href="#cite_note-1">[1]</a></sup>. Even after intensive and costly purification in wastewater treatment plants, these pollutants are still present in purified water and have so far been <b>unavoidably released and pollute our environment</b> <sup id="cite_ref-2"><a href="#cite_note-2">[2, </a></sup><sup id="cite_ref-3"><a href="#cite_note-3">3]</a></sup>! We want to tackle this problem and <b>support the fight against global pollution for a sustainable, clean and safe world</b>. <br> <br>
+<b>This inspired us to create B-TOX! </b><br> <br>
-One kind of micropollutants are pharmaceuticals, like diclofenac or azithromycin.  By design, they <b>evoke certain effects at low concentrations</b> and thus are very dangerous and detrimental for the environment in even very small amounts. For example, Diclofenac can cause <b>liver and kidney damage in fish</b>, at concentrations of <b>50 ng/L</b> <sup id="cite_ref-4"><a href="#cite_note-4">[4]</a></sup>. By that it is threatening whole fish populations. As we humans have produced these detrimental substances, it is <b>our responsibility to prevent their release</b> into the environment <sup id="cite_ref-5"><a href="#cite_note-5">[5]</a></sup>. Consequently, methods have to be applied that guarantee total degrading or rendering these substances nontoxic or at less toxic. <br> <br>
-<b>This inspired us to create our solution B-TOX! </b><br> <br>
 <div class="row">
@@ Line 117: / Line 115: @@
 </div><br><br>
-Our project B-TOX uses an <b>engineered modular <i>Bacillus&nbsp;subtilis</i> biofilm</b>, with enzymes displayed on the extracellular matrix. These enzymes can <b>neutralize many different micropollutants</b>. Our solution utilizes laccase enzymes, more specifically CueO and CotA, which we <b>immobilize in the extracellular matrix by fusing them to the major biofilm matrix component TasA</b>. Diclofenac is our initial target as it should be transformed into a less toxic substance by CueO and CotA <sup id="cite_ref-6"><a href="#cite_note-6">[6]</a></sup>. Besides from CueO and CotA we utilize the esterase EreB to reduce azithromycin in wastewater <sup id="cite_ref-7"><a href="#cite_note-7">[7]</a></sup>. Therefore, <b>B-TOX will reduce wastewater toxicity</b> and is a <b>contribution to the fight against global environmental pollution</b>. We consider our <b>cost-efficient approach</b> to be attractive for implementation in wastewater treatment plants <sup id="cite_ref-8"><a href="#cite_note-8">[8]</a></sup>. This may also <b>open doors for other biotechnological wastewater treatment projects</b> that can be combined with ours. Altogether we are convinced, that <b>B-TOX will bring the world one step closer in being sustainable.</b> <br> <br>
+B-TOX is an <b>engineered modular <i>Bacillus&nbsp;subtilis</i> biofilm</b>, with enzymes displayed on the extracellular matrix. These enzymes can <b>neutralize many different micropollutants</b>. Our solution utilizes laccase enzymes, more specifically CueO and CotA, which we <b>immobilize in the extracellular matrix</b> 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<sup id="cite_ref-6"><a href="#cite_note-6">[6]</a></sup>. Besides from CueO and CotA we utilize the esterase EreB to reduce azithromycin in wastewater<sup id="cite_ref-7"><a href="#cite_note-7">[7]</a></sup>. Therefore, <b>B-TOX will reduce wastewater toxicity</b> and is a contribution to the fight against global environmental pollution for a sustainable, clean and safe world.
+ <br> <br>
 <div class="referencestd">
@@ Line 137: / Line 136: @@
 <a class="anchor" id="cite_note-7"></a> <a class="referencestd" href=" https://pubs.acs.org/doi/10.1021/bi201790u " target="_blank"> [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. </a> <br>
-<a class="anchor" id="cite_note-8"></a> <a class="referencestd" href=" https://www.koms-bw.de/cms/content/media/2015_11_Kosten%20der%20Spurenstoffelimination%20auf%20Klaeranlagen%20-%20Erfahrungen%20aus%20Baden-Wuerttemberg_BWGZ.pdf " target="_blank"> [8] Dr. Steffen Metzger et al., Kosten der Spurenstoffelimination auf Kläranlagen - Erfahrungen aus Baden - Württemberg, BWGZ - Die Gemeinde, 2015, 11:549-553 </a> <br>
 </div>

Difference between revisions of "Team:TU Darmstadt/Poster"

Revision as of 17:30, 9 November 2020

Poster: TU_Darmstadt

References

References

CotA Modeling

EreB Modeling

Fusion Proteins modeling

Killswitch Modeling

Biofilm modeling

ABTS Assay

Kirby-Bauer Assay

Toxicity Assay

Flow Chamber

References

1) Introducing a kill switch

2) Engineering of B. subtilis strain

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

4) How to handle “B-TOX”

Awards: