Difference between revisions of "Part:BBa K3505025"
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | 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. | + | 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=== | ===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 | + | 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. |
− | [[Image:T--Thessaly--FFAR2-VTAIL-PHOT.png|900px|thumb|none|<I><b>Figure | + | [[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=== | ||
− | [[File:T--Thessaly--FFAR2-LACI-digestion.png|700px|thumb|none|<i><b>Fig. | + | [[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> | ||
+ | |||
+ | |||
+ | ===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. |
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.
Verification of Cloning
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1527
Illegal PstI site found at 2107 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1527
Illegal PstI site found at 2107 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1148
Illegal BamHI site found at 1349 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1527
Illegal PstI site found at 2107 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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.