Difference between revisions of "Part:BBa K2374006"
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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. | 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=== | ||
[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]] | [[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]] | ||
[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]] | [[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]] | ||
| Line 41: | Line 42: | ||
We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH. | We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH. | ||
| − | [[File:2017tongji image registry | + | [[File:2017tongji image registry 80test.png|center|400px|标题]] |
[http://2017.igem.org/Team:Tongji_China/Design More Information] | [http://2017.igem.org/Team:Tongji_China/Design More Information] | ||
Revision as of 17:48, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1453
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 479
Illegal BglII site found at 1075
Illegal BamHI site found at 137 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 487
Illegal BsaI site found at 533