Difference between revisions of "Part:BBa K2374006"
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===Design Note===
 
===Design Note===
  
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'''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.  
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We cloned synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pUAST-UAS-TH. The pUAST-UAS-TH also with the other two plasmids: pUAST-pleP-GAL4 ([https://parts.igem.org/Part:BBa_K2374005 BBa_K2374005])and pUAST-pleP-GAL80ts ([https://parts.igem.org/Part:BBa_K2374006 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. <br>The result of our testing on ''D.melanogaster'' is displayed below.
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[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br><br>
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[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br><br>
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[[File:2017tongji_image_registry_uTH.png|center|200px|pleP-GAL80ts]]  <br><br>
  
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[[File:2017tongji_image_registry_TH_base.png|center|300px|标题]]
 
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[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]] 
 
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[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]] 
 
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[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]] 
 
  
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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.
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Here shows the restriction endonuclease digestion image of pUAST-pleP-GAL80ts.
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So we recommend that you obtain from the constructed plasmid, or synthesize it directly.
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We ordered a synthetic '''ple''' from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.
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[[File:2017tongji_registry_image_P80.png|center|300px|标题]]
  
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[http://2017.igem.org/Team:Tongji_China/Design More Information]
  
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===Test Results===
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1. Use Real-time PCR to detect whether the expression of TH is increased at 29°C. It shows that the relative expression of TH in modified fruit flies increased significantly.<br>
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[[File:2017tongji_image_registry_qPCR.png|center|300px]]
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2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.<br>
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It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01]
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[[File:2017tongji_image_registry_behavior1.png|center|350px]]
  
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[http://2017.igem.org/Team:Tongji_China/Experiments More details]
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We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.
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[[File:2017tongji image registry 80test.png|center|400px|标题]]
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[http://2017.igem.org/Team:Tongji_China/Design More Information]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<partinfo>BBa_K2374006 parameters</partinfo>
 
<partinfo>BBa_K2374006 parameters</partinfo>
 
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===References ===
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1. Webster Nocholas, Jin Jiarui, Green Stephen, Hollis Melvyn, Chambon Pierre (1988). The Yeast UASG is a transcriptional enhancer in human hela cells in the presence of the GAL4 trans-activator. ''Cell''. 52 (2): 169–178.<br>
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2. West Jr. Robert W., Yocum R. Rogers, Ptashne Mark (1984). Saccharomyces cerevisiae GAL1-GAL10 Divergenet Promoter Region: Location and Function of the Upstream Activating Sequence UAS. ''Molecular and Cellular Biology''. 4 (11): 2467–2478.<br>
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3. Lewandoski Mark (2001). Conditional control of gene expression in the mouse. ''Nature Reviews Genetics''. 2: 743–755.

Latest revision as of 03:49, 2 November 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

We cloned synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pUAST-UAS-TH. The pUAST-UAS-TH also with the other two plasmids: pUAST-pleP-GAL4 (BBa_K2374005)and pUAST-pleP-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



Here shows the restriction endonuclease digestion image of pUAST-pleP-GAL80ts.

标题

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

Test Results

1. Use Real-time PCR to detect whether the expression of TH is increased at 29°C. It shows that the relative expression of TH in modified fruit flies increased significantly.

2017tongji image registry qPCR.png

2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.
It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01]

2017tongji image registry behavior1.png

[http://2017.igem.org/Team:Tongji_China/Experiments More details]

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


References

1. Webster Nocholas, Jin Jiarui, Green Stephen, Hollis Melvyn, Chambon Pierre (1988). The Yeast UASG is a transcriptional enhancer in human hela cells in the presence of the GAL4 trans-activator. Cell. 52 (2): 169–178.
2. West Jr. Robert W., Yocum R. Rogers, Ptashne Mark (1984). Saccharomyces cerevisiae GAL1-GAL10 Divergenet Promoter Region: Location and Function of the Upstream Activating Sequence UAS. Molecular and Cellular Biology. 4 (11): 2467–2478.
3. Lewandoski Mark (2001). Conditional control of gene expression in the mouse. Nature Reviews Genetics. 2: 743–755.