Composite

Part:BBa_K2374006

Designed by: Jianfeng Zhou   Group: iGEM17_Tongji_China   (2017-10-26)
Revision as of 20:30, 31 October 2017 by Amang Z (Talk | contribs) (→‎Design Note)


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.

标题


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 synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pSB1C3-UAS-TH and pUAST-UAS-TH. The pSB1C3-UAS-TH is for submission. The pUAST-UAS-TH also with the other two plasmids: pUAST-ple-GAL4 (BBa_K2374005)and pUAST-ple-GAL80ts (BBa_K2374006) are used to do micro-injection into the D.melanogaster. We must combine the three pathways to determine if the system work well. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.
The result of our testing on D.melanogaster is displayed below.

pleP-GAL4


pleP-GAL80ts


pleP-GAL80ts



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

标题

[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


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