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.
Team:TU Darmstadt/Poster
Poster: TU_Darmstadt
B-TOX: reduction of wastewater toxicity using a B. subtilis biofilm
Introduction
Micropollutants in wastewater are a global issue. These are anthropogenic substances like pharmaceuticals or chemical residues in general that pose an alarming threat to the environment and can even lead to the extinction of species. Various studies have been published that show concrete effects on the aquatic environment and therefore prove ecotoxicity among others [1]. In total the 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 want to tackle this problem and support the fight against global pollution for a sustainable, clean and safe world.
One kind of micropollutants are pharmaceuticals, like diclofenac or azithromycin. By design, they evoke certain effects at low concentrations and thus are very dangerous and detrimental for the environment in even very small amounts. For example, Diclofenac can cause liver and kidney damage in fish, at concentrations of 50 ng/L [4]. By that it is threatening whole fish populations. As we humans have produced these detrimental substances, it is our responsibility to prevent their release into the environment [5]. Consequently, methods have to be applied that guarantee total degrading or rendering these substances nontoxic or at less toxic.
This inspired us to create our solution B-TOX!
Our project B-TOX uses 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. We consider our cost-efficient approach to be attractive for implementation in wastewater treatment plants [8]. This may also open doors for other biotechnological wastewater treatment projects that can be combined with ours. Altogether we are convinced, that B-TOX will bring the world one step closer in being sustainable.
[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.
[8] Dr. Steffen Metzger et al., Kosten der Spurenstoffelimination auf Kläranlagen - Erfahrungen aus Baden - Württemberg, BWGZ - Die Gemeinde, 2015, 11:549-553
One kind of micropollutants are pharmaceuticals, like diclofenac or azithromycin. By design, they evoke certain effects at low concentrations and thus are very dangerous and detrimental for the environment in even very small amounts. For example, Diclofenac can cause liver and kidney damage in fish, at concentrations of 50 ng/L [4]. By that it is threatening whole fish populations. As we humans have produced these detrimental substances, it is our responsibility to prevent their release into the environment [5]. Consequently, methods have to be applied that guarantee total degrading or rendering these substances nontoxic or at less toxic.
This inspired us to create our solution B-TOX!
Our project B-TOX uses 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. We consider our cost-efficient approach to be attractive for implementation in wastewater treatment plants [8]. This may also open doors for other biotechnological wastewater treatment projects that can be combined with ours. Altogether we are convinced, that B-TOX will bring the world one step closer in being sustainable.
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.
[8] Dr. Steffen Metzger et al., Kosten der Spurenstoffelimination auf Kläranlagen - Erfahrungen aus Baden - Württemberg, BWGZ - Die Gemeinde, 2015, 11:549-553
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 an essential gene. It activates when B. subtilis leaves the biofilm. The inhibition in rpsB expression will trigger cell death due to the lack of rpsB hindering translation [1].
To achieve this, we replaced the native promoter of the rpsB gene with the degQ promoter (PdegQ)[2].
PdegQ is a quorum sensing (QS) controlled promoter and is activated by ComA-P. ComA-P is activated by the the QS signaling molecule ComX through a phosphorylation cascade [3]. QS signaling molecules are produced when cell density increases[4]. Therefore, concentration of signaling proteins increases with increasing cell density. Is cell density high enough concentration of signaling molecules is high and will keep the corresponding promoters active, like PdegQ. When cell density is low, low amounts of ComA-P are present and promoter activity is impaired.
In our case, an inactive PdegQ promoter should lead to lower expression levels of rpsB, resulting in cell death.
To prevent cell death at low cell densities we opted for a constitutive promoter in early stages of growth. When the right cell density is reached, and subsequently enough quorum sensing signaling peptides are present to sustain permanent induction of rpsB expression, the constitutive promoter is exchanged. PdegQ is already a target for the QS regulator protein ComA-P [5]. Consequently, we decided to design a cassette including the constitutive Pveg promoter for the growth phase and the QS activated PdegQ promoter for implementation (see Fig. 1).
Figure 1: our kill switch cassette
In order to replace Pveg with PdegQ, both are flanked by the mutated cre-recombination sites lox66 and lox71. These sites can be recognized by the Cre-recombinase. If the cre recombinase is present, it induces the inversion of the sequence in between the recombination sites. As thereby the lox66 is mutated it can no longer be recognized after inversion making this procedure irreversible [6].
The cre recombinase will be integrated into the genome of B. subtilis under control of a xylose induced promoter. For that we use the iGEM part BBa_K733002[7].
In conclusion, our cassette will replace the native rpsB promoter. To this end, we integrate our cassette directly upstream of the gene utilizing homologous recombination. B. subtilis integrates the cassette into its genome by adding 500 bp homologous flanks upstream and downstream of our cassette[8].
To verify the safety and functionality of our kill switch, we planned some experiments to verify and furtherly improve our kill switch. A very important change would be the usage of an inducible promoter for the growth phase, instead of the constitutive Pveg promoter. We planned on using the PcymRC maryonette promoter, induced by cuminic acid[9]. After the cassette is inverted during cre recombinase induction, we do no longer induce this promoter. Any cells that did not invert the cassette and therefor exchange the promoter with the quorum sensing activated one will die, as rpsB is no longer produced.
Figure 2: our improved kill switch cassette
In addition, we will add a GFP coding sequence on the upstream antisense strand (See fig. 2). After invertion, the inducible promoter now lies in front of the GFP gene. When adding inducer to the cells, they will produce GFP which can be easily confirmed, even by eyesight. That way, even wastewater treatment plant worker can easily verify, whether the cells have a functioning kill switch.
[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). https://link.springer.com/article/10.1007/BF00333661
[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, http://www.subtiwiki.uni-goettingen.de/v3/gene/view/40C1E81BAB04BD1CC98FC57DF25D27DBDFEB5A59
[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, https://doi.org/10.1016/j.tim.2004.11.007 https://www.cell.com/trends/microbiology/fulltext/S0966-842X(04)00261-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0966842X04002616%3Fshowall%3Dtrue
[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). https://doi.org/10.1038/s41589-018-0168-3
To achieve this, we replaced the native promoter of the rpsB gene with the degQ promoter (PdegQ)[2].
PdegQ is a quorum sensing (QS) controlled promoter and is activated by ComA-P. ComA-P is activated by the the QS signaling molecule ComX through a phosphorylation cascade [3]. QS signaling molecules are produced when cell density increases[4]. Therefore, concentration of signaling proteins increases with increasing cell density. Is cell density high enough concentration of signaling molecules is high and will keep the corresponding promoters active, like PdegQ. When cell density is low, low amounts of ComA-P are present and promoter activity is impaired.
In our case, an inactive PdegQ promoter should lead to lower expression levels of rpsB, resulting in cell death.
To prevent cell death at low cell densities we opted for a constitutive promoter in early stages of growth. When the right cell density is reached, and subsequently enough quorum sensing signaling peptides are present to sustain permanent induction of rpsB expression, the constitutive promoter is exchanged. PdegQ is already a target for the QS regulator protein ComA-P [5]. Consequently, we decided to design a cassette including the constitutive Pveg promoter for the growth phase and the QS activated PdegQ promoter for implementation (see Fig. 1).
Figure 1: our kill switch cassette
In order to replace Pveg with PdegQ, both are flanked by the mutated cre-recombination sites lox66 and lox71. These sites can be recognized by the Cre-recombinase. If the cre recombinase is present, it induces the inversion of the sequence in between the recombination sites. As thereby the lox66 is mutated it can no longer be recognized after inversion making this procedure irreversible [6].
The cre recombinase will be integrated into the genome of B. subtilis under control of a xylose induced promoter. For that we use the iGEM part BBa_K733002[7].
In conclusion, our cassette will replace the native rpsB promoter. To this end, we integrate our cassette directly upstream of the gene utilizing homologous recombination. B. subtilis integrates the cassette into its genome by adding 500 bp homologous flanks upstream and downstream of our cassette[8].
To verify the safety and functionality of our kill switch, we planned some experiments to verify and furtherly improve our kill switch. A very important change would be the usage of an inducible promoter for the growth phase, instead of the constitutive Pveg promoter. We planned on using the PcymRC maryonette promoter, induced by cuminic acid[9]. After the cassette is inverted during cre recombinase induction, we do no longer induce this promoter. Any cells that did not invert the cassette and therefor exchange the promoter with the quorum sensing activated one will die, as rpsB is no longer produced.
In addition, we will add a GFP coding sequence on the upstream antisense strand (See fig. 2). After invertion, the inducible promoter now lies in front of the GFP gene. When adding inducer to the cells, they will produce GFP which can be easily confirmed, even by eyesight. That way, even wastewater treatment plant worker can easily verify, whether the cells have a functioning 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). https://link.springer.com/article/10.1007/BF00333661
[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, http://www.subtiwiki.uni-goettingen.de/v3/gene/view/40C1E81BAB04BD1CC98FC57DF25D27DBDFEB5A59
[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, https://doi.org/10.1016/j.tim.2004.11.007 https://www.cell.com/trends/microbiology/fulltext/S0966-842X(04)00261-6?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0966842X04002616%3Fshowall%3Dtrue
[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). https://doi.org/10.1038/s41589-018-0168-3
Modeling
Because there was no prior knowledge about the binding properties of diclofenac and CotA,
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. 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
To evaluate our structures, we took a look at the Ramachandran plot. Most dihedral angles laid in allowed positions giving a first validations of our predicted structure. For further control, we ran an MD simulation.
The results showed that the structures are indeed stable.
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.
Modeling reaction networks can give a deeper insight into the workings of the reaction network. Therefore, we decided to model the kill switch reaction pathways. To get a plausible result, we needed to assume the following assumptions:
The pandemic presented us with a major problem. We were not able to carry out any experimental work.
So we came together with iGEM Hannover to build a software able to simulate biofilm growth.
The result was quit fascinating. We used physical laws to model bacteria growth and interactions
and probability theory to model biological phenomenon.
The source code was written in python and is highly modular, so you can expand the model as you wish.
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
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. 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 structuresTo evaluate our structures, we took a look at the Ramachandran plot. Most dihedral angles laid in allowed positions giving a first validations of our predicted structure. For further control, we ran an MD simulation.
The results showed that the structures are indeed stable.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.
Modeling reaction networks can give a deeper insight into the workings of the reaction network. Therefore, we decided to model the kill switch reaction pathways. To get a plausible result, we needed to assume 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.
$$1. \frac {d(ComX)}{dt} = [Tunnel Protein] - ComP$$
$$2. \frac {d(PrecomX)}{dt} = -ComQ$$
$$3. \frac {d(ComX1)}{dt} = ComQ - [Tunnel Protein]$$
$$4. \frac {d(ComA)}{dt} = -ComP$$
$$5. \frac {d([ComA+P])}{dt} = ComP - [Binding/Unbinding]$$
$$6. \frac {d(DNA)}{dt} = [Binding/Unbinding]$$
$$7. \frac {d(mRNA)}{dt} = Transkription - [mRNA Degradation]$$
$$8. \frac {d(Protein)}{dt} = Translation - [Protein Degradation]$$
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:
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.
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.
- 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.
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:
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.
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
Our developed kill switch will ensure that our bacteria will not be able to survive without a biofilm due to dependence on quorum sensing. Before our biofilm will perform quorum sensing, we will induce cell growth and biofilm formation separately in the laboratory. When "B-TOX" is ready and the necessary cell density is reached it can be put into the clarifier.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…
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:
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.
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
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: 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
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