Difference between revisions of "Part:BBa K3866012"

 
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<partinfo>BBa_K3866012 short</partinfo>
 
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===Usage and Biology===
 
===Usage and Biology===
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This composite part constitutes the G-protein coupled bioreceptor composite of the dual TANGO-GPCR assay(Dogra, Sona, Kumar and Yadav, 2016) (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)(Kaemmerer, 2010)  . 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.
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When TEV protease cleaves , the lac repressor  is released and binds to the lac operator . In the presence of SCFAs the GPCR is activated.
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===Design Notes===
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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 p3 omega1R vector and has overhangs compatible for GoldenBraid cloning.
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[[Image:T--Thessaly--FR.png|900px|thumb|none|<I><b>Figure 1.</b> The level Ω module of GPCR-Tango Module. FFAR-RRAA </i>]]
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===Verification of Cloning===
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[[File:T--Thessaly--FRgel.png|700px|thumb|none|<i><b>Fig.2:</b>: (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)</i>]]
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===Exerimental Use and Experience===
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Thsi part was used at <bbpart>BBa_K3866014</bbpart>
  
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3866012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3866012 SequenceAndFeatures</partinfo>
  
  
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===References===
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===Functional Parameters===
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*Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.
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<partinfo>BBa_K3866012 parameters</partinfo>
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*Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.
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Revision as of 13:06, 4 October 2021


ParaBAD:RBS-FFAR2:AVPR2 tail:TCS-LacI-terminator---ParaBAD-RraA-Terminator

Usage and Biology

This composite part constitutes the G-protein coupled bioreceptor composite of the dual TANGO-GPCR assay(Dogra, Sona, Kumar and Yadav, 2016) (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)(Kaemmerer, 2010) . 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 p3 omega1R vector and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level Ω module of GPCR-Tango Module. FFAR-RRAA

Verification of Cloning

Fig.2:: (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)

Exerimental Use and Experience

Thsi part was used at BBa_K3866014

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 3028
    Illegal BamHI site found at 3229
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3407
    Illegal PstI site found at 3987
    Illegal NgoMIV site found at 3487
    Illegal AgeI site found at 983
    Illegal AgeI site found at 2863
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI site found at 2845
    Illegal SapI site found at 3683
    Illegal SapI site found at 3938
    Illegal SapI.rc site found at 3140


References

  • Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.
  • Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.