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
Introduce your project and your team's goals.
Pharmies
We were concentrating on the problematic substances diclofenac (analgetic) and azithromycin (antibiotic). Both substances pose environmental hazards in high concentrations. They reach our wastewater in large quantities. At wastewater treatment plants, only a small part can be degraded endangering aquatic fauna and flora. Other substances, such as ibuprofen or estrogen are also problematic.
We utilize enzymes that can modify such substances rendering them less toxic. We chose the two laccases CueO (E. coli ) and CotA ( B. Subtilis ) for diclofenac, as well as the esterase EreB ( E. coli ) for antibiotics like erythromycin and azithromycin. Laccases are oxidoreductases that can oxidize phenolic compounds like in diclofenac, as well as other pharmaceuticals, such as carbamazepine, 17β-Estradiol. The resulting products are demonstrably less toxic. EreB has already been shown to being capable of breaking down erythromycin effectively. EreB also displays some promiscuity towards azithromycin. To increase the activity of EreB towards azithromycin we want to utilize site-directed mutagenesis.
With the selected enzymes we are already able to degrade a wide range of pharmaceuticals. Besides the already mentioned substances, chloramphenicol, bisphenol A and 4-nonylphenol can also be oxidized by laccase.
Enabling us to adapt our biofilm to different local conditions of wastewater pollution. Because of this B-TOX is not restricted to one area.
Biofilm
Where is the biofilm? Why is the Biofilm? How is the Biofilm
Killswitch
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
To replace Pveg with PdegQ they are flanked by the mutated cre-recombination sites lox66 and lox71. These sites can be recognized by Cre-recombinase. When cre recombinase is present, it is able to induce the inversion of the sequence in between the recombination sites. As 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 do so, we integrate our cassette directly upstream of the gene. For this we plan oan utilizing homologous recombination. By adding 500 bp homologous flanks upstream and downstream of our cassette, b subtilis integrates the cassette into its genome[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 key aspect of our project is displaying degradation enzymes in the biofilm matrix by fusing them to the major biofilm matrix component TasA. Therefore, a critical issue for our project is biofilm stability. For every new enzyme we want to display in the biofilm, we have to prove that TasA is still functional and able to connect Bacillus subtilis cells to provide a stable biofilm. Aditionally, we have to make sure that no cells escape into the environment when being exposed to the rough conditions that prevail in a wastewater treatment plant (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 WWTP. 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. Since biofilm stability is an essential issue for more iGEM teams we created a manual and contribute the files for 3D-printing to the iGEM community. Our approach is a low cost and presents a 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 – as in 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 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.
Not only the survey, also the mail address we established for the audience of our podcast allows us to get in direct contact with the society.
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.
Furthermore, we did a livestream together with iGEM Kaiserslautern and hosted a zoom call for students from a school near Darmstadt.
- 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: With professors of philosophy and a member of the ethic commission of our university we talked 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
some WWTP stuff i guess
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: (medal criteria)
Bronze
Gold
Best Education
We started a publicly accessible survey covering the opinion and knowledge about SynBio within Germany. Based on this survey, we broadcasted a livestream with iGEM Kaiserslautern to enable an open dialogue and interact with people all over Germany. We provided information to people who sought more knowledge and talked to them about their concerns regarding water pollution.
In our German podcast “Genomenal - Ein Haufen Zellen redet über Biotech” (English: Genomenal – a bunch of cells talk about biotech), we address ethical and legal questions in biotechnology. We provided our listeners with ways to easily communicate their thoughts to us and included their feedback. Additionally, we created a “how to podcast” guideline for anyone with the same intention covering necessities like equipment needs and how to build up your own RSS-feed.
We programmed a jump-and-run computer game called “The Genomal Adventures of Dr. W” together with Aleksa Zečević to playfully spark interest in biology even among the youngest of our audience.
The renowned German scientific journal BIOspektrum published our article, enabling us to reach an audience of 15,000 monthly readers. The BIOspektrum is linked to many German Societies in science such as GBM, VAAM, GfG or DPGT. As a result of this publication, Prof. Dr. Möller, astromicrobiologist at the German Aerospace Center, reached out to us to talk about possible implementation scenarios of our biofilm in aerospace and the adjustments necessary to enable its usage e.g. aboard the international space station (ISS).
Best Integrated Human Practices
We reached out to experts like Dr. Ulrich Ehlers from the Federal Office for Consumer Protection and Food Safety who also suggested the use of a kill switch to enable a safer use of our project.
As Prof. Dr. Sibylle Gaisser from the department of industrial biotechnology in Ansbach recommended, we created a safety form with the goal to prevent misuse of our biofilm, as well as a safety sheet to educate employees of wastewater treatment plants on biosafety issues.
We talked to many experts about all aspects of our project and used the information they provided us with to make our project come to maturation.
Best Model
We developed a software to model biofilm growth in collaboration with iGEM Hannover. It incorporates e.g. growth rates, split length or movement speeds of bacteria, enabling us to create plots of their position and movement as well as the robustness of the biofilm. It is based on literature values although experimental data can be easily integrated.
We used MATLAB to model our quorum sensing-based kill switch mechanism using ordinary differential equations (ODE) to make predictions about its functionality. It is also based on literature values with the intention to incorporate experimental data as soon as possible.
We determined possible 3D structures for one of our degradation enzymes EreB using RosettaCM. Using GROMACS for molecular dynamics simulation (MD), we validated the stability of our enzyme structure. With RosettaCM, we also 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. One of which is the “bacteria” class which is an object-orientated representation of the bacteria in the biofilm. Secondly, there is the biofilm class, which simulates interactions forces using a biophysical potential, and drag force and finally the “constants” class, which represents the interface the user utilizes to specify simulation constants like duration, step size or output paths. Other constants like initial population size and bacterial strain (Escherichia coli or Bacillus subtilis) may also be chosen by the user while the addition of one’s own parameters allows for easy simulation of one's bacterial population of interest. 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. To ensure access to water and sanitation for all, especially, as in target 6.3, to improve water quality by reducing pollution and minimizing the release of hazardous chemicals is part of goal 6 and an important part of our project. We designed this biofilm for the purpose of wastewater treatment without complex and costly technological adjustments but with the versatility of bacterial biofilms. We can see the consequences of releasing pharmaceutical pollutants into the environment as it has been reported to endanger several species in the recent past. That is why goal 14 inspires us to conserve and sustainable use the oceans, seas and marine resources and to significantly reduce marine pollution as covered in target 14.1. It is of great importance to us to prevent harm to our marine ecosystems due to contamination of the environment with pharmaceutical pollutants.
Bronze
- Competition Deliverables: We registered for the virtual Giant Jamboree and are looking forward to the event! Therefore, we created our Wiki, Poster, Presentation Video, Project Promotion Video and filled in the Judging Form.
- Attributions: Please visit our Acknowledgements to see who supported us this year.
- Project Description: We present ourproject description on our Wiki and a short version can be seen at “Introduction”.
- Contribution: We contributed a variety of things to the iGEM community this year. Click on “Contributions” to see.
- Engineering Success: We carefully designed our parts and show how to test them and evaluate results.
- Collaboration: We collaborated with different teams throughout the year, like thepostcard collaboration with Duesseldorf, iJET with Aachen, Oxiteers with Stuttgart and Kaiserslautern and many more.
- Human Practices: We engaged with experts and the public during the course of our project. We were able to generate a lot of valuable input and insights.
- Proposed Implementation: We explain how our project will be implemented and give an outlook on alternative implementations, e.g. aboard the ISS.
Gold
- Integrated Human Practices: We spoke to different groups of interests and ages, addressing their worries and incorporating their ideas in our project design. Especially, regarding the biosafety aspect of our project.
- Project Modeling: We modelled our enzymes and fusion enzymes, biofilm growth and formation, and our kill switch. Together with iGEM Hannover we developed a software tool to model biofilm formation.
- Partnership: We engaged in a partnership called the Oxiteers, with iGEM Stuttgart and iGEM Kaiserslautern.
- Science Communication: We developed several strategies to educate society and make our efforts accessible for everyone.
Awards:
Best Education
We started a publicly accessible survey covering the opinion and knowledge about SynBio within Germany. Based on this survey, we broadcasted a livestream with iGEM Kaiserslautern to enable an open dialogue and interact with people all over Germany. We provided information to people who sought more knowledge and talked to them about their concerns regarding water pollution.
In our German podcast “Genomenal - Ein Haufen Zellen redet über Biotech” (English: Genomenal – a bunch of cells talk about biotech), we address ethical and legal questions in biotechnology. We provided our listeners with ways to easily communicate their thoughts to us and included their feedback. Additionally, we created a “how to podcast” guideline for anyone with the same intention covering necessities like equipment needs and how to build up your own RSS-feed.
We programmed a jump-and-run computer game called “The Genomal Adventures of Dr. W” together with Aleksa Zečević to playfully spark interest in biology even among the youngest of our audience.
The renowned German scientific journal BIOspektrum published our article, enabling us to reach an audience of 15,000 monthly readers. The BIOspektrum is linked to many German Societies in science such as GBM, VAAM, GfG or DPGT. As a result of this publication, Prof. Dr. Möller, astromicrobiologist at the German Aerospace Center, reached out to us to talk about possible implementation scenarios of our biofilm in aerospace and the adjustments necessary to enable its usage e.g. aboard the international space station (ISS).
Best Integrated Human Practices
We reached out to experts like Dr. Ulrich Ehlers from the Federal Office for Consumer Protection and Food Safety who also suggested the use of a kill switch to enable a safer use of our project.
As Prof. Dr. Sibylle Gaisser from the department of industrial biotechnology in Ansbach recommended, we created a safety form with the goal to prevent misuse of our biofilm, as well as a safety sheet to educate employees of wastewater treatment plants on biosafety issues.
We talked to many experts about all aspects of our project and used the information they provided us with to make our project come to maturation.
Best Model
We developed a software to model biofilm growth in collaboration with iGEM Hannover. It incorporates e.g. growth rates, split length or movement speeds of bacteria, enabling us to create plots of their position and movement as well as the robustness of the biofilm. It is based on literature values although experimental data can be easily integrated.
We used MATLAB to model our quorum sensing-based kill switch mechanism using ordinary differential equations (ODE) to make predictions about its functionality. It is also based on literature values with the intention to incorporate experimental data as soon as possible.
We determined possible 3D structures for one of our degradation enzymes EreB using RosettaCM. Using GROMACS for molecular dynamics simulation (MD), we validated the stability of our enzyme structure. With RosettaCM, we also 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. One of which is the “bacteria” class which is an object-orientated representation of the bacteria in the biofilm. Secondly, there is the biofilm class, which simulates interactions forces using a biophysical potential, and drag force and finally the “constants” class, which represents the interface the user utilizes to specify simulation constants like duration, step size or output paths. Other constants like initial population size and bacterial strain (Escherichia coli or Bacillus subtilis) may also be chosen by the user while the addition of one’s own parameters allows for easy simulation of one's bacterial population of interest. 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. To ensure access to water and sanitation for all, especially, as in target 6.3, to improve water quality by reducing pollution and minimizing the release of hazardous chemicals is part of goal 6 and an important part of our project. We designed this biofilm for the purpose of wastewater treatment without complex and costly technological adjustments but with the versatility of bacterial biofilms. We can see the consequences of releasing pharmaceutical pollutants into the environment as it has been reported to endanger several species in the recent past. That is why goal 14 inspires us to conserve and sustainable use the oceans, seas and marine resources and to significantly reduce marine pollution as covered in target 14.1. It is of great importance to us to prevent harm to our marine ecosystems due to contamination of the environment with pharmaceutical pollutants.
Outlook
Use this section to explain whatever you would like! Suggestions: Outlook
Acknowledgements
Here you can see all the great people who helped us during this year.
Primary PI: Prof. Warzecha
Secondary PI: Prof. Kabisch
Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, Leon Kraus, Chris Sürder and Maximilian Zander
Primary PI: Prof. Warzecha
Secondary PI: Prof. Kabisch
Our advisors: Fran Bacic Toplek, Sebastian Barthel, Simone Bartl-Zimmermann, Alexander Gräwe, Leon Kraus, Chris Sürder and Maximilian Zander
- Others:
- Dr. Dietz,
- Angelina Folberth,
- Dr. Immel,
- Jörg Kalkowski,
- PD Dr. Kletzin,
- Dr. Mikosch-Wersching,
- Prof. Schmidt,
- Prof. Stein,
- Prof. Waldminghaus,
- Barbara Wolf,
- Prof. van der Vegt,
- Aleksa Zecevic,
- Working group of Prof. Kabisch and Prof. Warzecha,
- People from „Krautnah“ and from Hda of TU Darmstadt
- Former iGEMers
- Klara Eisenhauer,
- Jonathan Funk,
- Peter Gockel,
- Jamina Gerhardus,
- Robin Johannson,
- Thea Lotz,
- Tim Maier,
- Benjamin Meyer,
- Jean Victor Orth,
- Hannah Rainer,
- Lara Steinel,
- Leon Werner,
- Marius Wollrab
- Experts
- Dipl. Ing. Udo Bäuerle,
- PhD Yunrong Chai,
- Dr. Ulrich Ehlers,
- Prof. Dr. Sibylle Gaisser,
- Florian Heyn,
- Wolfgang John,
- Prof. Jürgens,
- Prof. Lackner,
- Prof. Dr. Ralf Möller,
- Prof. Nordmann,
- Prof. Dr. Jörg Oehlmann,
- Dr. Sabine Sané,
- Dr. Dietmar Schlosser,
- Dr. Patrick Schröder,
- Thomas Seeger,
- Prof. Dr. Jörg Stülke
Sponsors