Part:BBa_K2374003

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.

Design Notes

ple has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.

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

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

A pUAST-UAS-TH
B pUAST-UAS-TH BglII

We also did one site direct mutagenesis for submission:
Pst I (647) CTGCAG->CTGCTG

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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 13
  Illegal PstI site found at 256
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 13
  Illegal PstI site found at 256
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1540
  Illegal XhoI site found at 672
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 13
  Illegal PstI site found at 256
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 13
  Illegal PstI site found at 256
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 918
  Illegal BsaI site found at 1884
  Illegal BsaI.rc site found at 636
  Illegal BsaI.rc site found at 1402
  Illegal BsaI.rc site found at 1551