Difference between revisions of "Part:BBa K3505026"

Revision as of 17:56, 27 October 2020

ParaBAD:RBS- β-arrestin2:TEVp:terminator

Usage and Biology

The TANGO GPCR-assay, takes advantage of the b-arrestin capabilities to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease. Upon SCFAs-induced activation of the FFAR2 receptor, β-arrestin 2 is recruited and binds to the intracellular part of the receptor. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) and subsequently release lac repressor. Then, the lac repressor is free to inhibit expression of downstream elements of our signaling system, by binding to a lac operator sequence.

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 alpha2 vector BBa_K3505009 and has overhangs compatible for Golden Braid cloning.

Figure 2. The level B module of GPCR-Tango module : ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator

Verification of Cloning

Fig.1:: (U=Uncut , C= Cut) Restriction digestion of ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator (C1-C 4), with :HindIII (C1a-C4a), Expected bands : 2847+2490+719 bp , EcoRV (C1b-C4b) , Expected bands:3988 bp+2214 bp. Positive result: C1,C2,C3,C4. (C1a and C1b -same sample etc)

Sequence and Features