Difference between revisions of "Part:BBa K2374001"
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We cloned this 452bp TH promoter easily from ''D. melanogaster'' 's genomic DNA and the sequencing result is correct.
 
We cloned this 452bp TH promoter easily from ''D. melanogaster'' 's genomic DNA and the sequencing result is correct.
  
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We connected TH promoter to GAL4( BBa_K2374004 [https://parts.igem.org/Part:BBa_K2374004] )and GAL80ts( BBa_K2374002 [https://parts.igem.org/Part:BBa_K2374002] )respectively, and cloned them into pUAST vector which had been removed the UAS sequence( BBa_K2374008 [https://parts.igem.org/Part:BBa_K2374008] ), then to microinject them into ''Drosophila'' 's eggs. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.
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We connected TH promoter to GAL4( BBa_K2374004 [https://parts.igem.org/Part:BBa_K2374004] )and GAL80ts( BBa_K2374002 [https://parts.igem.org/Part:BBa_K2374002] )respectively, and cloned them into pUAST vector which had been removed the UAS sequence( BBa_K2374008 [https://parts.igem.org/Part:BBa_K2374008] ), then to microinject them into ''Drosophila'' 's eggs. Also we did microinjection with UAS-TH (BBa_K2374003 [https://parts.igem.org/Part:BBa_K2374003]). After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system. Here shows some results.
 
[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br> <br> <br>  
 
[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br> <br> <br>  
 
[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br>
 
[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br>
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<center>
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<video controls width="700">
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    <source src="https://static.igem.org/mediawiki/2015/2/2a/China-Tongji-Project-video4-pmyo2-chR2.mp4" type="video/mp4" />pmyo2-chR2.mp4
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</video>
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</center>
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We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.  
 
We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.  
 
[[File:2017tongji image registry ptest.png|center|400px|标题]]
 
[[File:2017tongji image registry ptest.png|center|400px|标题]]

Revision as of 18:26, 30 October 2017


TH (ple) promoter-> (fruit fly)

Overview

TH (ple) is a tyrosine hydroxylase, the first and rate-limiting step in the synthesis of dopamine (and eventually, melanin). Dopamine has critical roles in system development. This part is a 452bp sequence in 5' upstream of the ple exon. TH promoter is activated in the substantia nigra densities, ventral tegmental area, hypothalamus, olfactory bulb, norepinephrine and adrenergic neurons of the brain, also in the sympathetic ganglia and adrenal gland medulla and chromaffin cells.


Design Notes

We get this 452bp sequence from FlyBase ( Dmel\ple [http://flybase.org/reports/FBgn0005626.html]). We regard the 5'-UTR region as promoter region of ple.
This should not be the most accurate and concise promoter sequence. We hope that subsequent users will be able to accurately identify the promoter sequence.

We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells.
We cloned this 452bp TH promoter easily from D. melanogaster 's genomic DNA and the sequencing result is correct.

We connected TH promoter to GAL4( BBa_K2374004 [1] )and GAL80ts( BBa_K2374002 [2] )respectively, and cloned them into pUAST vector which had been removed the UAS sequence( BBa_K2374008 [3] ), then to microinject them into Drosophila 's eggs. Also we did microinjection with UAS-TH (BBa_K2374003 [4]). After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system. Here shows some results.

pleP-GAL4



pleP-GAL80ts

<video controls width="700"> 

   <source src="https://static.igem.org/mediawiki/2015/2/2a/China-Tongji-Project-video4-pmyo2-chR2.mp4" type="video/mp4" />pmyo2-chR2.mp4

</video>

We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.

标题

We did 2 mutagenesis on this sequence.
site direct mutagenesis:
1. EcoR I (184) GAATTC->GATTTC
2. Xba I (219) TCTAGA->TGTAGA

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 137
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Source

GeneCards®: The Human Gene Database

NCBI

FlyBase

References

Harrington CA, Lewis EJ, Krzemien D, Chikaraishi DM. Identification and cell type specificity of the tyrosine hydroxylase gene promoter. Nucleic Acids Research. 1987;15(5):2363-2384.