Difference between revisions of "Part:BBa K3505025"
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<partinfo>BBa_K3505025 short</partinfo> | <partinfo>BBa_K3505025 short</partinfo> | ||
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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(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> | ||
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+ | ===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.