Composite

Part:BBa_K3505025

Designed by: Asteria Tsapadikou,Emmanouil Stylianakis   Group: iGEM20_Thessaly   (2020-10-20)
Revision as of 18:51, 27 October 2020 by Asteriat (Talk | contribs) (→‎Usage and Biology)


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 (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). 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. Sequence and Features

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 Golden Braid cloning.

Figure 2. The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator

Verification of Cloning

Fig.1:: (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
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Parameters
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