As the first representatives of Hungary at a collegiate level, we were very excited to join the iGEM community. For us, the first months were constant learning as we deep-dived into the world of the competition. We learned about the Parts library, distribution kits, wiki pages, etc. And as our project was maturing from a faint idea into a complex and well-designed work, we broadened the iGEM community’s tools and contributed with ideas for future teams.
Parts registry
We contributed to the parts library with a new blue light activatable system: BLADE (BBa_K4375004), based on Romano et al. [1]. This system uses AraC binding proteins fused to a Vivid domain that dimerizes upon blue light illumination. Aside from the already existing pDawn system (BBa_K1616019) now, there is a new part in the registry that can be utilized for future teams. We also developed and registered devices where either a mCherry or a cytotoxic Cytolisin-A (BBa_K811000) is the coding region behind the AraC promoter (BBa_K4375021 and BBa_K4375019 respectively). As optogenetics is a highly improving research area, we believe these systems will have an important part in the iGEM competition.
Aside from BLADE, we also broadened the parts registry with two nanobodies (BBa_K4375005 and BBa_K4375006). These proteins play a significant role in synthetic biology as they are small, suitable for bacterial expression, and because of their size they easily penetrate solid tumors [2]. We contributed to the registry with CEA and EGFR antigen-specific nanobodies as basic parts and as well as composite part devices when they are attached to an Intimin part, which was also our contribution to the library (BBa_K4375016, BBa_K4375017, and BBa_K4375012). This Intimin enables the attachment of the Nanobody to the outer membrane of the bacterial cell wall, thus making our bacteria tumor-specific [3]. The importance of these elements can be described by the role they are playing in the development of synthetic biology and the fact that these proteins are already used in several devices. Future iGEM teams now can use these constructs to build their own NanoBody display systems, or just simply be creative with the nanobodies.
We also registered a new tag, a small fluorescent protein, miRFP670nano as a basic part (BBa_K4375008). This was fused to an INPNC part, which we improved by codon optimization as an already existing part (BBa_K4375007). This was registered as an improved part (BBa_K4375018). The linker between was designed to include a BamHI restriction site (BBa_K4375009). This was also registered, as well as another linker derived from llama, providing aid for nanobody construct design for future teams (BBa_K4375011). Finally, we added a c-Myc-derived Myc tag and its variant, one that contains a SpHI cut site (BBa_K4375014 and BBa_K4375013).
To summarize: we added a total number of 17 new parts!
- 11 basic
- 4 composite
- 2 improved
These can be utilized by future teams, as they are compatible with most of the Assembly techniques, including BioBrick and MoClo. We are proud of our contribution to the community and hope these parts will serve the next generation of teams well!
Hardware
One of the most important things we can add to the community is the hardware we build. This system allows blue-light activatable systems to be tested. Our first design, Instrument I. is capable of supporting illumination for Petri-dishes as well as flasks and also provides cooling by a ventilator. We designed this to be affordable, easy to build, and in case of malfunction, the parts can be exchanged easily. We prioritized these qualities as we pursued a system that is accessible and, therefore, useful for future teams who wish to build a system similar to ours but do not necessarily have the options for it.
The second design, RaccOpto, was an improvement on the first one after many considerations. This system is built for 96-well plates, is controllable, and is similar to the previous one: affordable. This was again emphasized. These designs are perfect starting points for anyone who wishes to construct a hardware to test biological systems.
We provided the building elements list for both of the designs and a brief description of the most important elements. These can be found on our Hardware site.
Documentation
As we browsed the parts registry for information on components we wished to use in our project, we often overcome scarce information reporting on them. This prompted us to read the literature, and we found some new properties of them. Cytolysin-A is a core component of our project design, as this pore-forming protein is responsible for the killing of the tumor cells upon its expression activated by blue light. This protein is already registered in the iGEM Parts Registry. However, while we searched the properties of this protein, we encountered the fact that there is little information provided on the details of its mechanism. Since it is very important for us to understand the basics of the elements used in our project, we dived into the literature.
One thing we found was the fact that its pore-forming can be enhanced with the addition of cholesterol to the measurements. The other important note we found was that it leaves the bacteria in OMVs. The details can be found on the Parts page.
Surveys
As the project went along, we considered it important to measure the general attitude of the people in our community. That is why we constructed a survey to question people about their viewpoint on GMOs and synthetic biology or whether they would participate in therapy like our project. The answers and the structure of the survey can be found on our Survey Form page.
Education and communication
During our project, we also emphasized the importance of spreading the concepts of synthetic biology among laics. After we concluded from our surveys that often people in Hungary are not familiar with the basics of this topic even though it has ever-growing importance in our everyday life, we decided to participate in this year's European Researcher’s Night. This event was designed by us to educate the visitors from the basics of biology to the concepts of synthetic biology. Our exhibited models and experiments were made to be easily reproducible, and most of them were DIY. We explained to them our project and the importance of synthetic biology through examples and games (More information on the Communication page). After this event, we even visited high schoolers to give them an even deeper explanation and to tell them about specifically optogenetics, synthetic biology-based therapies, and solutions we need to come up with. (More information on the Education page)
Inclusivity
Finally, we contributed to the community with an inclusive approach. In our country, science is often out of reach, so this is something we could understand. That is why we considered it important to have our video supported with subtitles translated into 11 languages (English, Hungarian, French, Spanish, Italian, German, Slovak, Serbian, Croatian, Russian, and Arabic). These are often ones spoken by many people, like English, Arabic, Russian, Spanish, German, or French, but we also considered it significant to have languages accessible to smaller countries. This is often neglected in many places and could set up boundaries between people and science. That is why we included the languages of countries around Hungary, like Serbia, Croatia, and Slovakia. We also wanted to make our idea accessible to the hearing-impaired, which is why we added sign language to our video. (Watch our videos on the Inclusivity page)
[1] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nat. Chem. Biol. 2021, 17, 817–827. https://doi.org/10.1038/s41589-021-00787-6.
[2] Chen, W.; Yuan, Y.; Jiang, X. Antibody and Antibody Fragments for Cancer Immunotherapy. J. Controlled Release 2020, 328, 395–406. https://doi.org/10.1016/j.jconrel.2020.08.021.
[3] Piñero-Lambea, C.; Bodelón, G.; Fernández-Periáñez, R.; Cuesta, A. M.; Álvarez-Vallina, L.; Fernández, L. Á. Programming Controlled Adhesion of E. Coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins. ACS Synth. Biol. 2014, 4, 463–473. https://doi.org/10.1021/sb500252a.