Contribution

Contribution

We designed a dose dependent response system based on the NarX-NarL two component nitrate sensing system of Escherichia coli . This design combines the continuous expression of a NarX mutant protein that is unable to transduce the signal to its secondary messenger, NarL and the inducible expression of the wild type protein NarX from the naringenin responsive promoter designed by De Peape et. al., (2018). The next contribution is the partial characterization of a temperature responsive kill switch based on RNA thermometers. Lastly, we worked on a document that enables future teams to plan out the competition, the challenges and the deadlines. This is based solely on our own experiences and has the goal of offering some idea of what is coming. This idea arose when we met the new team from the VUB (Brussels, Belgium) and a researcher at Antwerp University (Belgium) who mentioned his interest in starting an iGEM team, but needed some input on how to do so.

Naringenin responsive promoter, kill switch and new teams


Future IGEM Teams

Our team has made multiple contributions valuable for future iGEM teams in different areas of the competition. First, we worked on the naringenin responsive promoter developed by De Paepe et al., (2018). We believe that any biosensor should be capable of detecting different concentrations of the biomarker it is designed to detect. This arose from the rationale that many biomarkers indicate an environmentally dangerous condition only at certain concentrations, or as applied in our project, a disease. Therefore, we developed a system that does not respond up until a certain concentration and when it reaches a threshold, the biosensor gives the desired response. This response could be in the shape of a signal such as fluorescence or as a therapeutic. To achieve this, we needed one promoter that constitutively expresses at a certain rate while the second promoter is inducible and shows a rather linear response curve. The research group of Marjan De Mey (Ghent University, Belgium) developed such a promoter and was very interested in discussing their paper. After some meetings, we decided to make use of the design called pSynSense2.5000 (part BBa_K4345017). In their research, De Paepe et al. (2018) designed different ribosomal binding sites in vitro and tested the responses to different concentrations of naringenin. For a more detailed description of the research, we happily refer you to their paper. The contribution here is thus the addition of this part to the iGEM registry. On the other hand, we designed this part to be both a dose-detector and a dose-respondent using the NarX-NarL two-component system.



Kill Switches

Secondly, we worked on robust kill switches based on RNA thermometers. RNA thermometers are natural thermoregulators present in the 5’-untranslated region (UTR) of heat shock proteins. We’ve tested and partially characterized the kill switches and registered their composite parts under BBa_K4345020, BBa_K4345023 and BBa_K4345024. Lastly, we made a contribution not related to scientific design or experimental results. Our teams at KU Leuven (Belgium) start each year with limited info on how the competition will go. This caused some confusion to us, which motivated us to work on a document for next year's team. In this document, we describe the workflow we went through, the people we contacted to help with regulatory issues, practicalities and other general information. After meeting the iGEM team from VUB (Brussels, Belgium), they mentioned being a new team includes similar challenges. Lastly, we spoke to a researcher at the university of Antwerp (Belgium) who is very interested in starting an iGEM team to compete in next year’s competition. Therefore, we modified the script we made for next year's KU Leuven team towards a general guide for new teams.