Difference between revisions of "Part:BBa K3505025"

Revision as of 19:15, 27 October 2020

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. 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 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