Difference between revisions of "Part:BBa K3505026"
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<partinfo>BBa_K3505026 short</partinfo> | <partinfo>BBa_K3505026 short</partinfo> | ||
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===Usage and Biology=== | ===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=== | ===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 <bbpart>BBa_K3505009</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 alpha2 vector <bbpart>BBa_K3505009</bbpart> and has overhangs compatible for GoldenBraid cloning. |
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+ | [[Image:T--Thessaly--B-ARRESTIN-TEV-PHOT.png|900px|thumb|none|<I><b>Figure 1.</b> The level B module of GPCR-Tango module : ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator</i>]] | ||
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+ | ===Verification of Cloning=== | ||
+ | [[File:T--Thessaly--B-ARRESTIN-TEV-digestion.png|700px|thumb|none|<i><b>Fig.2:</b>: (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)</i>]] | ||
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===Sequence and Features=== | ===Sequence and Features=== | ||
<partinfo>BBa_K3505026 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3505026 SequenceAndFeatures</partinfo> |
Latest revision as of 00:37, 28 October 2020
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.
Verification of Cloning
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 965
Illegal SapI.rc site found at 2788
Illegal SapI.rc site found at 3136