Difference between revisions of "Part:BBa K3866014"

Revision as of 13:32, 4 October 2021

TANGO module NOT GATE SCFAs detection

Usage and Biology

This composite part constitutes the G-protein coupled bioreceptor composite of the dual TANGO-GPCR assay(Dogra, Sona, Kumar and Yadav, 2016) (the other part is the b-arrestin-2 constituent, placed under the control of the arabinosed-induced promoter. FFAR2 is a naturally found eukaryotic GPCR protein that exhibits high affinity for SCFAs (acetic, propionic and butyric acid)(Kaemmerer, 2010) . After background searching through the NCBI database, we have identified the gene coding sequences needed for the designing of this gene construct. More specifically, a vasopressin receptor 2 segment has been attached to the C-terminal of the receptor, as it mediates recruitment of a TEV tagged b-arrestin-2, along with a TEV cleavage site. When TEV protease cleaves , the lac repressor is released and binds to the lac operator . In the presence of SCFAs the GPCR is activated.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in seva alpha1R vector BBa_K3505008and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level a final module of GPCR-Tango Module.

Verification of Cloning

Fig.2:: (U=Uncut , C= Cut) Restriction digestion a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminato (C1a-C4b) with : BamHI(C1a-C4a) , Expected bands : 2847+2225 bp , EcoRV (C2a-C2b) ,Expected bands : 3587 bp + 2845 bp, Positive result: C1,C2,C3,C3 (C1a and C1b is the same sample etc)

Exerimental Use and Experience

This TANGO NOT GATE after SCFAs induction should lower the Fluorecence.

Cultures grown in LB and induced with 1% L-arabinose for 3 hours. t=0 is considered when SCFAs are added.

The positive control is the TU with ECFP only BBa_K3505032 The negative controls are the empty vector BBa_K3505008 with and without SCFAs. The lines in between are TANGO modules with 0, 0,1, 1 and 10 mM Sodium Acetate

Conclusion

We observe that there was continuous non-specific suppression, both in the presence and absence of SCFAs. We hypothesize that 1. either suppression takes place without the release of the LacI from the GPCR before it reaches the membrane, or 2. the TEV protease cleaves the LacI non-specifically.

Sequence and Features

References

• Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.
• Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.