Difference between revisions of "Part:BBa K3505025"

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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 (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.
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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.
 
When TEV protease cleaves , the lac repressor  is released and binds to the lac operator . In the presence of SCFAs the GPCR is activated.
 
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===
 
===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 <bbpart>BBa_K3505008</bbpart> and has overhangs compatible for Golden Braid cloning.
+
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 <bbpart>BBa_K3505008</bbpart> and has overhangs compatible for GoldenBraid cloning.
  
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[[Image:T--Thessaly--FFAR2-VTAIL-PHOT.png|900px|thumb|none|<I><b>Figure 2.</b> The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator </i>]]
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[[Image:T--Thessaly--FFAR2-VTAIL-PHOT.png|900px|thumb|none|<I><b>Figure 1.</b> The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator </i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--FFAR2-LACI-digestion.png|700px|thumb|none|<i><b>Fig.1:</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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[[File:T--Thessaly--FFAR2-LACI-digestion.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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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3505025 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3505025 SequenceAndFeatures</partinfo>
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 +
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===References===
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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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*Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.

Latest revision as of 00:34, 28 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(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 alpha1R vector BBa_K3505008 and has overhangs compatible for GoldenBraid cloning.

Figure 1. The level B module of GPCR-Tango Module : a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator

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)



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 1349
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1527
    Illegal PstI site found at 2107
    Illegal NgoMIV site found at 1607
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965
    Illegal SapI site found at 1803
    Illegal SapI site found at 2058
    Illegal SapI.rc site found at 1260


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.