Difference between revisions of "Part:BBa K2374003"
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K2374007 short</partinfo>
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<partinfo>BBa_K2374003 short</partinfo>
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
! colspan="2" style="background:#FFBF00;"|TH
 
! colspan="2" style="background:#FFBF00;"|TH
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[[File:2017tongji_registry_image_dopa.png|center|600px|dopa]]  
 
[[File:2017tongji_registry_image_dopa.png|center|600px|dopa]]  
 
<br>
 
<br>
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 +
===Design Notes===
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'''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.
 +
 +
[[File:2017tongji_image_registry_TH_base.png|center|300px|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 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>
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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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We cloned TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-TH.
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[[File:2017tongji image registry UASTHtest.png|center|400px|标题]]
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site direct mutagenesis for submission::<br>
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1. EcoR I (130) GAATTC->AAATTC <br>
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2. Pst I (782) CTGCAG->CTGCTG
 +
 +
===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>
 +
[[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]
 +
===Source===
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 +
GeneCards®: The Human Gene Database [http://www.genecards.org/cgi-bin/carddisp.pl?gene=TH]
 +
 +
NCBI [https://www.ncbi.nlm.nih.gov/gene/?term=38746]
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 +
FlyBase [http://flybase.org/reports/FBgn0005626.html]
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===References===
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 +
Janice A. Fischer, et al. GAL4 activates transcription in ''Drosophila''. ''Nature'' 332, 853 - 856 (1988)
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2374007 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2374003 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2374007 parameters</partinfo>
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<partinfo>BBa_K2374003 parameters</partinfo>
 
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Latest revision as of 03:08, 2 November 2017


ple (Tyrosine 3-monooxygenase, TH) -> (fruit fly)

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

ple (Tyrosine 3-monooxygenase, TH)

ple (TH) is a rate-limiting enzyme in the dopamine’s synthesis, and it plays an important role in the physiology of adrenergic neurons.
Catalytic activity: L-tyrosine + tetrahydrobiopterin + O2 = L-dopa + 4a-hydroxytetrahydrobiopterin.
This subpathway is part of the pathway dopamine biosynthesis, which is itself part of catecholamine biosynthesis.


dopa


Design Notes

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

标题

site direct mutagenesis for submission::
1. EcoR I (130) GAATTC->AAATTC
2. Pst I (782) CTGCAG->CTGCTG

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]

Source

GeneCards®: The Human Gene Database [http://www.genecards.org/cgi-bin/carddisp.pl?gene=TH]

NCBI [5]

FlyBase [http://flybase.org/reports/FBgn0005626.html]

References

Janice A. Fischer, et al. GAL4 activates transcription in Drosophila. Nature 332, 853 - 856 (1988)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1157
    Illegal XhoI site found at 289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 535
    Illegal BsaI site found at 1501
    Illegal BsaI.rc site found at 253
    Illegal BsaI.rc site found at 1019
    Illegal BsaI.rc site found at 1168