Difference between revisions of "Part:BBa K2374006"

Revision as of 17:49, 31 October 2017

TH-Gal80ts

TH-GAL80ts
Use in	D.melanogaster
RFC standard	RFC 10 compatible
Backbone	pSB1C3
Submitted by	[http://2017.igem.org/Team:Tongji_China Tongji_China 2017]

Overview

A dimer of GAL80 binds to the C-terminal ends of the GAL4 dimer so that, while it can still bind to a UAS sequence, it can no longer activate transcription. This interaction of GAL4 and GAL80 can be taken advantage of to reﬁne the expression pattern of GAL4-dependent transgenes.
We use the specific promoter pleP (TH Promoter) to control the fixed expression of GAL80ts, because of the specificity of pleP, GAL80ts express in tissue which express dopamine specifically. At 25℃, GAL4 and GAL80ts express, GAL80tsp conbine with GAL4p then stop it to bind to UAS, so the TH do not express.At 29℃, GAL80ts is inactivated, which cannot combine with GAL4p, so GAL4p binds to UAS and starts the expression of TH, leading to the high expression of dopamine.

Design Note

ple has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.

According to our experiment results to judge, the ple coding sequence is hard to clone from Drosophila 's cDNA library because of its multi-segment repeats. So we recommend that you obtain from the constructed plasmid, or synthesize it directly.

We ordered a synthetic ple from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.

We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.

[http://2017.igem.org/Team:Tongji_China/Design More Information]

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1453
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 479
Illegal BglII site found at 1075
Illegal BamHI site found at 137
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 487
Illegal BsaI site found at 533

@@ Line 24: / Line 24: @@
 ===Design Note===
-[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]]
-[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
-[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
 '''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.
 [[File:2017tongji_image_registry_TH_base.png|center|300px|标题]]
+[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]]
+[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
+[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
 According to our experiment results to judge, the '''ple''' coding sequence is hard to clone from ''Drosophila'' 's cDNA library because of its multi-segment repeats.