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Team:Evry/AIDSystem
From 2012.igem.org
Intertissue communication: An orthogonal hormonal system
We adapted the auxin production device from the iGEM team Imperial college 2011 to eukaryotes and combined it with an auxin detection module. This way, we created the first synthetic hormonal system for inter-tissues communication.
To test this system, we co-injected plasmids expressing our production and reception devices in embryos, a new chassis we wanted to implement for synthetic biology. We performed auxin toxicity and uptake tests at the begining of our project to ensure the feasability.
Auxin production devices
We designed three auxin production devices in embryos. The devices 1 and 2 were designed to be expressed in embryos, while device 3 was designed to be expressed in E. coli. In this last case, the aim is that the tadpoles eat bacteria expressing device 3.
- Auxin production device 1 : this device is composed of BBa_K812021, coding for IaaM, and BBa_K812120, coding for IaaH for auxin generator for the use in embryos.
- Auxin production device 2 : this device is composed of BBa_K812014. It is meant for the co-expression of IaaH and IaaM genes in the same cells in embryos.
- Auxin production device 3 : this device is composed of BBa_K515100, coding for IaaM and IaaH for auxin generator in E.coli.
Pathway
Auxin reception devices
Our reception system is based on the auxin-degron system established by K. Nishimura and all. (2009). This system allows a rapid depletion of protein in nonplant cells.
We designed two auxin production devices in embryos. To visualize the communication between different tissues or between E. coli and a tissue of the embryo, we chose to work with GFP. Our orthogonal hormonal system works with any proteins fused to AID signal with any transcription factors.
- Auxin reception device 1 : this device is composed of BBa_K812010 coding for GFP-AID, and BBa_K812012 coding for OsTir1.
- Auxin reception device 2 : this device is composed of BBa_K812013 coding for GFP-AID and OsTir1 in the same cell.
Auxin degron system
Auxin binds osTir1 and promotes the interraction of E3 ubiquitin which recruits E2. This mechanism allows the polyubiquitination and the adressage of the protein to the proteasome.
To ensure this system, we checked that Xenopus tropicalis possesses the Skp1, Cul1 and Rbx1 genes.
Conclusion
The implementation of a tissue communication underlines the interest for the use of eucaryotes such as Xenopus in synthetic biology. This tool could be used with any protein fused with AID signal, allowing to test it in a multitissular system.
Our orthogonal hormonal system was tested in pCS2+ plasmid. We have imagined to insert I-Sce sites in this plasmid to ensure its integration in the chromosome. So far, creating a durable tissue communication system.
References:
- 1. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature methods 6, 917-22 (2009).
