Difference between revisions of "Part:BBa K3505026"

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.

Figure 1. The level B module of GPCR-Tango module : ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator

Verification of Cloning

Fig.2:: (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)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633
21
INCOMPATIBLE 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
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633
25
INCOMPATIBLE 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
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 965
Illegal SapI.rc site found at 2788
Illegal SapI.rc site found at 3136

@@ Line 6: / Line 6: @@
 ===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) .
@@ Line 12: / Line 13: @@
-[[Image:T--Thessaly--B-ARRESTIN-TEV-PHOT.png|900px|thumb|none|<I><b>Figure 2.</b> The level B module of GPCR-Tango module : ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator</i>]]
+[[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>]]
 ===Verification of Cloning===
-[[File:T--Thessaly--B-ARRESTIN-TEV-digestion.png|700px|thumb|none|<i><b>Fig.1:</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>]]
+[[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>]]