Team:TU Darmstadt/Contribution
As in every iGEM year contributions to the iGEM community are a big part of every project. Due to COVID-19 we were not able to test any of our designed parts in the lab. Nevertheless, we provided basic parts together with literature and information for future teams to characterize.
In addition, we provide the iGEM community with several other contributions ranging from How-Tos, a self-designed flow chamber to our partnership page which gives insights into wastewater treatment with laccases for new teams.
In addition, we provide the iGEM community with several other contributions ranging from How-Tos, a self-designed flow chamber to our partnership page which gives insights into wastewater treatment with laccases for new teams.
Flow Chamber
A critical issue for projects involving bacterial biofilms is the mechanical stability. Determination of biofilm stability is especially important for our project as we plan on displaying degradation enzymes in the biofilm matrix by fusing them to TasA. For every new enzyme we want to display in the biofilm, we have to prove that TasA is still functional and able to connect B. subtilis cells to provide a stable biofilm. In addition, we have to consider biofilm stability since our kill switch would be triggered if cell density is too low through biofilm disintegration. Investigating this matter, we designed a flow chamber for comparing different bacterial strains (e.g. different B. subtilis strains) regarding their biofilm stability. Therefore, flow rates can be adjusted by the experimentator. Our flow chamber is specifically designed for pumping liquids in a thin layer over biofilm samples. Thereby, not only enabling us to analyse and measure the amount of living material that is washed off the biofilm, but also allowing to design new experiments and assays by simply changing the method of analysis.
We developed a manual and contribute the files for 3D printing of the flow chamber parts for future iGEM teams. They can utilize the flow chamber for their own experiments and will be able to fit the analysis method to their need. Examples for this might be testing the washout of immobilized enzymes or the resilience towards mechanical forces.
Firstly, the flow chamber has to be manufactured which requires a 3D printer. Consequently, our flow chamber is attractive to the whole iGEM community due to the low cost. Based on that another advantage is the simplicity in adapting our system. The bottom part contains a notch in which (in our case) a biofilm can be grown. Additionally, other samples, e.g. the mesoporous silica foam with immobilized laccase from our friends of the iGEM team Stuttgart, can be inserted in the notch and therefore tested for mechanical stability. In a nutshell our flow chamber is a low cost and highly modular system which can be useful for various applications. We are happy to contribute such a valuable and easily accessible idea in shape of this convenient and functional system that conceals so much potential for future iGEM teams.
The Oxiteers
Our “Oxiteers”-partnership with the iGEM teams Stuttgart and Kaiserslautern is
aimed towards making it easier for future iGEM teams to tackle the issue of wastewater treatment.
Since this topic has become quite popular over the course of the years, e.g. iGEM Bielefeld in 2012 or iGEM Stockholm in 2018,
and our teams are working on fighting the diclofenac pollution in German wastewater this season, we decided to team up.
Therefore, we compared our projects, alongside with our workflows,
and came up with a subpage filled with experts regarding wastewater (treatment) and literature on this topic. Here you can find our joint wikipage.
Part Contribution
In addition to further characterization of already existing parts we also contribute 10 basic parts and one composite part. You can find our contributed modelling results on the following parts: BBa_K3429011, BBa_K3429012, BBa_K3429013 here.
EreB
We examined the properties of the erythromycin esterase type II EreB (BBa_K1159000), that already shows promiscuous activity towards azithromycin, with macromolecular computational modeling approaches using the Rosetta and GROMACS applications. The crystal structure of EreB is not yet solved e.g. by analysis techniques like X-ray diffraction or Cryo-EM. Therefore, we applied a homology modeling approach on the EreB sequence using Rosetta’s comparative modeling tool RosettaCM. When blasting the protein sequence against the Protein Data Bank (PDB) we found a succinoglycan biosynthesis protein possessing high structural similarity in its active site to EreB. We were able to find a candidate for EreB crystal structure after generating 20,000 structures and validated its structural stability by molecular dynamics simulation (MD). In a 100 ns simulation we were able to prove structural stability especially in EreB’s active site by principal component (PCA), Root-mean-square deviation (RMSD), small root-mean-square fluctuation (RMSF) and radii of gyration (Rg) analysis. To prove structural stability of the fusion protein of EreB with B. Subtilis extracellular matrix protein TasA (BBa_K3429013) that we want to utilize for enzyme immobilization we predicted the fusion protein’s structure by comparative modeling and observed structural stability by MD simulation, that was again analyzed by PCA, RMSD, RMSF and Rg. In conclusion, we were able to generate a 3D structure candidate for EreB and validate our method of protein immobilization with TasA.
Recombinases
As we want to use the Cre recombinase derived from the P1 bacteriophage for our kill switch, we conducted some further literature research in order to contribute to further application of the Cre recombinase. See Part:BBa_K1680007.
How to B-TOX
We made a manual for employees of wastewater treatment plants (WWTP), in which we give instructions on how to handle our product “B-TOX“.
In there, not only the risks of GMOs are described, but also a safety protocol and the application of “B-TOX” in the WWTP are to be found.
For further information please click here.
How to Rosetta
The Rosetta Commons Software developed by Baker Lab is a powerful computational toolbox for the modeling and analysis of protein structures. It shines with the broad applicability and variability of its applications. The problem most people face with the software is that it is not very forgiving towards beginners and not particulary convenient to use in general. We ourselves faced a lot of difficulties with our modeling and are hoping to make the lives of future iGEMers just a little bit easier by providing a guide to the applications that we used for our project and adding in our own tipps and tricks.
How to Podcast - A guide for the iGEM community
This year we created a podcast about biotechnology. There are many aspects to be considered turning an idea into a final podcast. This is why we decided to give the iGEM community an overview about everything they need to think about during the creation process and what we ourselves have learned. For further information please click here.