Difference between revisions of "Part:BBa K2374003"

Latest revision as of 03:08, 2 November 2017

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

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_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].

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

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.

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]

[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