Composite

Part:BBa_K3505026

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


ParaBAD:RBS- β-arrestin2:TEVp: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 alpha2 vector BBa_K3505009 and has overhangs compatible for GoldenBraid cloning.


Figure 1. The level B module of GPCR-Tango module : ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator

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)



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 2854
    Illegal PstI site found at 1546
    Illegal PstI site found at 1633
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 2854
    Illegal PstI site found at 1546
    Illegal PstI site found at 1633
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1446
    Illegal BamHI site found at 1148
    Illegal XhoI site found at 1843
    Illegal XhoI site found at 2237
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 2854
    Illegal PstI site found at 1546
    Illegal PstI site found at 1633
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 2854
    Illegal PstI site found at 1546
    Illegal PstI site found at 1633
    Illegal AgeI site found at 983
    Illegal AgeI site found at 1829
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI.rc site found at 2788
    Illegal SapI.rc site found at 3136
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Categories
Parameters
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