Difference between revisions of "Part:BBa K2374003"

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.

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

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

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K2374003 short</partinfo>
-===Overview===
 {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
-! colspan="2" style="background:#FFBF00;"|UAS-TH
+! colspan="2" style="background:#FFBF00;"|TH
 |-
 |'''Use in'''
@@ Line 19: / Line 17: @@
 |[http://2017.igem.org/Team:Tongji_China Tongji_China 2017]
 |}
+'''ple''' (Tyrosine 3-monooxygenase, TH)
-An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription, the UAS is often considered to be analogous to the function of the enhancer in multicellular eukaryotes. Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. The UAS is found adjacently upstream to a minimal promoter (TATA box) and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin.
+'''ple''' (TH) is a rate-limiting enzyme in the dopamine’s synthesis, and it plays an important role in the physiology of adrenergic neurons.<br>
+'''Catalytic activity''':
+L-tyrosine + tetrahydrobiopterin + O2 = L-dopa + 4a-hydroxytetrahydrobiopterin.<br>
+This subpathway is part of the pathway dopamine biosynthesis, which is itself part of catecholamine biosynthesis.<br>
-Gene '''ple''' encodes Tyrosine 3-monooxygenase which also known as TH ([https://enzyme.expasy.org/EC/1.14.16.2 EC:1.14.16.2]).
+[[File:2017tongji_registry_image_dopa.png|center|600px|dopa]]
-It plays an important role in the physiology of adrenergic neurons. This protein is involved in step 1 of the subpathway that synthesizes dopamine from L-tyrosine. Dopamine has critical roles in system development. Proteins known to be involved in the 2 steps of the subpathway in this organism are: <br>
+<br>
-.Tyrosine 3-monooxygenase ('''ple''') <br>
-.no protein annotated in this organism <br>
-This subpathway is part of the pathway dopamine biosynthesis, which is itself part of Catecholamine biosynthesis.
-It belongs to the biopterin-dependent aromatic amino acid hydroxylase family. We also provide '''ple''' promoter in [https://parts.igem.org/Part:BBa_K2374001 BBa_K2374001].
+===Design Notes===
-===Design notes===
-[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]]
-[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
-[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
 '''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.
@@ Line 45: / Line 38: @@
 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].
+[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]   <br> <br>
+[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]   <br><br>
+[[File:2017tongji_image_registry_uTH.png|center|200px|pleP-GAL80ts]]   <br><br>
+We cloned TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-TH.
-We cloned synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pSB1C3-UAS-TH and pUAST-UAS-TH. The pSB1C3-UAS-TH is for submission. The pUAST-UAS-TH also with the other two plasmids: pUAST-ple-GAL4 ([https://parts.igem.org/Part:BBa_K2374005 BBa_K2374005])and pUAST-ple-GAL80ts ([https://parts.igem.org/Part:BBa_K2374006 BBa_K2374006]) are used to do micro-injection into the ''D.melanogaster''. We must combine the three pathways to determine if the system work well. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system. <br>The result of our testing on ''D.melanogaster'' is displayed below.
-[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]]
-[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
-[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
-'''''note''''':
-. At 18-25°C (the optimum temperature for fruit flies’ growth), it has the activity binding to Gal4, which will eliminate the effect Gal4 binding to UAS, then downstream gene TH will not express and the expression level of dopamine is normal.<br>
-. When the temperature is up to 29℃, Gal80ts will be inactivated, then Gal4 works properly, binding to Gal4 binding sequence on UAS, and start the expression of downstream gene TH which will leads to the overexpression of dopamine in ''Drosophila''.<br>
-[http://2017.igem.org/Team:Tongji_China/Design More Information]
-We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.
 [[File:2017tongji image registry UASTHtest.png|center|400px|标题]]
-[http://2017.igem.org/Team:Tongji_China/Design More Information]
+site direct mutagenesis for submission::<br>
+. EcoR I (130) GAATTC->AAATTC <br>
+. Pst I (782) CTGCAG->CTGCTG
 ===Test Results===
@@ Line 74: / Line 59: @@
 [http://2017.igem.org/Team:Tongji_China/Experiments More details]
-===Usage and Biology===
-The protein encoded by this gene is involved in the conversion of tyrosine to dopamine. It is the rate-limiting enzyme in the synthesis of catecholamines, hence plays a key role in the physiology of adrenergic neurons. Mutations in this gene have been associated with autosomal recessive Segawa syndrome. Alternatively spliced transcript variants encoding different isoforms have been noted for this gene.
-<!-- -->
-<span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K2374003 SequenceAndFeatures</partinfo>
-===Functional Parameters===
-TH is involved in step 1 of the subpathway that synthesizes dopamine from L-tyrosine and plays an important role in the physiology of adrenergic neurons.
 ===Source===
@@ Line 99: / Line 67: @@
 FlyBase [http://flybase.org/reports/FBgn0005626.html]
+===References===
+Janice A. Fischer, et al. GAL4 activates transcription in ''Drosophila''. ''Nature'' 332, 853 - 856 (1988)
+<!-- Add more about the biology of this part here
+===Usage and Biology===
+<!-- -->
+<span class='h3bb'>Sequence and Features</span>
+<partinfo>BBa_K2374003 SequenceAndFeatures</partinfo>
+<!-- Uncomment this to enable Functional Parameter display
+===Functional Parameters===
 <partinfo>BBa_K2374003 parameters</partinfo>
 <!-- -->
-===References ===
-. Webster Nocholas, Jin Jiarui, Green Stephen, Hollis Melvyn, Chambon Pierre (1988). The Yeast UASG is a transcriptional enhancer in human hela cells in the presence of the GAL4 trans-activator. ''Cell''. 52 (2): 169–178.<br>
-. West Jr. Robert W., Yocum R. Rogers, Ptashne Mark (1984). Saccharomyces cerevisiae GAL1-GAL10 Divergenet Promoter Region: Location and Function of the Upstream Activating Sequence UAS. ''Molecular and Cellular Biology''. 4 (11): 2467–2478.<br>
-. Lewandoski Mark (2001). Conditional control of gene expression in the mouse. ''Nature Reviews Genetics''. 2: 743–755.