Composite

Part:BBa_K3505025

Designed by: Asteria Tsapadikou,Emmanouil Stylianakis   Group: iGEM20_Thessaly   (2020-10-20)


ParaBAD:RBS-FFAR2:AVPR2 tail:TCS-LacI-terminator


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 alpha1R vector BBa_K3505008 and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator

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)



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 1349
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
    Illegal NgoMIV site found at 1607
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI site found at 1803
    Illegal SapI site found at 2058
    Illegal SapI.rc site found at 1260


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.
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