Difference between revisions of "Part:BBa K2374001"
 
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===Overview===
 
===Overview===
  
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|ple promoter
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|-
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|'''Use in'''
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|''D.melanogaster''
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10] compatible
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|-
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|'''Backbone'''
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|pSB1C3<br>
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|-
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|'''Submitted by'''
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|[http://2017.igem.org/Team:Tongji_China Tongji_China 2017]
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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.
 
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.  
 
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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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. 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.
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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_K2374007 [https://parts.igem.org/Part:BBa_K2374007]). 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[http://2017.igem.org/Team:Tongji_China/Experiments].
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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_ple4.png|center|200px|pleP-GAL4]]  <br> <br>
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[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <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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<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>
 
  
 
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]]
 
We did 2 mutagenesis on this sequence.<br>
 
We did 2 mutagenesis on this sequence.<br>
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site direct mutagenesis:<br>
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site direct mutagenesis for submission::<br>
 
1. EcoR I (184)  GAATTC->GATTTC <br>
 
1. EcoR I (184)  GAATTC->GATTTC <br>
 
2. Xba I  (219)  TCTAGA->TGTAGA
 
2. Xba I  (219)  TCTAGA->TGTAGA
 +
 +
===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.<br>
 +
[[File:2017tongji_image_registry_qPCR.png|center|300px]]
 +
2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.<br>
 +
It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01]
 +
[[File:2017tongji_image_registry_behavior1.png|center|350px]]
 +
 +
[http://2017.igem.org/Team:Tongji_China/Experiments More details]
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:55, 2 November 2017


TH (ple) promoter-> (fruit fly)

Overview

ple promoter
Use in D.melanogaster
RFC standard RFC 10 compatible
Backbone pSB1C3
Submitted by [http://2017.igem.org/Team:Tongji_China Tongji_China 2017]

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_K2374007 [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[http://2017.igem.org/Team:Tongji_China/Experiments].

pleP-GAL4


pleP-GAL80ts


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 for submission::
1. EcoR I (184) GAATTC->GATTTC
2. Xba I (219) TCTAGA->TGTAGA

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
    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.