From 2012.igem.org

Latest revision as of 22:37, 26 October 2012

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.

Auxin is a molecule of choice for working on tadpole. It is amphiphilic and hydrophobic so we assume it can cross the biological membrane easily but its toxicity is reported to be very weak. It can be synthetized in a two step pathway from a tryprophane precursor.

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.

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

One key requirement for the creation of a synthetic hormonal system was to find a cytosolic hormone receptor. We came across a very ingenious system developped my Masato Kanemaki laboratory coming from rice and that has been reported to work and patented in mammalian cells but not in tadpoles.

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

This system is based on the E3 ubiquitinase SCF-TIR1, that is capable, in the presence of auxin to recognized GFP tagged with AID degron. When auxin is present in the cytosol, TIR1 binds to the degron and recruits the E2 ubiquitinase that adds the ubiquitine tag. Then, the GFP is degraded by the proteasome, giving a decrease in the fluorescence of the tissue.

To ensure this system works, we checked that Xenopus tropicalis possesses the Skp1, Cul1 and Rbx1 genes.

Example: Skin-Kidney communication

The choice of tissues has been set on the specific properties of the tissues in term of function and blod irrigation as well as on the existance of reported functionnig tissue specific promotors. As an emitter, we chose to use the skin, and as a receiver, the kidney.

In order to trigger the emission of the hormone in our synthetic hormonal system we wanted to dissolve a chemical in the water of the tadpole that would activate an indicible promoter. The most exposed tissue to the chemical environment is undoubtely the epithelium, because of the important surface exposed to the water. On the top of it, this tissue is highly vascularized, which is important in order to acheive a high concentration of auxin in the blood. An important library of promoter has also been identified for this tissue.

The problem of introducing a non native hormon into the blood is that the kidney is likely to eliminate it from the blood. The kidney works as an inverted filter, in the sense that it takes out every molecule from the blood and reintroduce only the one it knows, and our hormon does not nessarily belongs to these molecule. Therefore, we can anticipate that the course of your molecule will ends up there and it will be the place where it is the most concentrated. This organ seems to be the best place for expressing our receiver system. There are also good promoters coming from the different ion channels that are know to work there.

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. Using I-Sce sites, we could insert this system onto the chromosome. This would creating a durable tissue communication system for longer term use.