Difference between revisions of "Part:BBa K3866013"

Revision as of 13:46, 4 October 2021

ParaBAD:RBS- β-arrestin2:TEVp:terminator---pAndersonJ23115:lacO:RBS-eCFP -terminator

Usage and Biology

This part has an inducible promoter from arabinose, leading to a regulated expression of the transcription unit. B-arrestin has the ability 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.TEV protease recognizes and cleaves the cleavage site. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) .

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 omega2 vector BBa_K3505011and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level Ω module of GPCR-Tango module : B-arrestin -TEV protease- ECFP

Verification of Cloning

Fig.2:: (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)

Experimental Use and Experience

This part was used in BBa_K3866014

Sequence and Features