Difference between revisions of "Part:BBa K2374001"
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<partinfo>BBa_K2374001 short</partinfo>
 
<partinfo>BBa_K2374001 short</partinfo>
  
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Pale 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.
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===Overview===
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This promoter starts in cell which express dopamine specifically.
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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.
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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.
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===Design Notes===
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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'''.<br>
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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.<br><br>
 +
 
 +
We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells. <br>
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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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[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br> <br> <br>
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[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br>
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We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
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[[File:2017tongji image registry ptest.png|center|400px|标题]]
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We did 2 mutagenesis on this sequence.<br>
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site direct mutagenesis:<br>
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1. EcoR I (184)  GAATTC->GATTTC <br>
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2. Xba I  (219)  TCTAGA->TGTAGA
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===Source===
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GeneCards®: The Human Gene Database
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NCBI
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FlyBase
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===References===
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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.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:09, 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. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.

pleP-GAL4



pleP-GAL80ts

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

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.

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]