Difference between revisions of "Part:BBa K2374003"

Revision as of 20:56, 30 October 2017

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

Overview

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

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.

Gene ple encodes Tyrosine 3-monooxygenase which also known as TH (EC:1.14.16.2). 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:
1.Tyrosine 3-monooxygenase (ple)
2.no protein annotated in this organism
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 BBa_K2374001.

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

We cloned UAS-TH into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.

标题

[http://2017.igem.org/Team:Tongji_China/Design More Information]

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]

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. 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

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

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

NCBI [1]

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

References

1. 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.
2. 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.
3. Lewandoski Mark (2001). Conditional control of gene expression in the mouse. Nature Reviews Genetics. 2: 743–755.

@@ Line 29: / Line 29: @@
 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===
@@ Line 34: / Line 36: @@
 [[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
 [[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
-GAL80/GAL80ts is a important part of UAS/GAL4 system to achieve gene expression in specific cells. <br>
-We cloned this 1311bp GAL80ts sequence easily from ''D. melanogaster'' (Gifts by Chunyang Ni)'s genomic DNA and the sequencing result is correct.
-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[http://2017.igem.org/Team:Tongji_China/Experiments].
+'''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.
-[[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_TH_base.png|center|300px|标题]]
-[[File:2017tongji_image_registry_uTH.png|center|200px|pleP-GAL80ts]]   <br><br>
+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 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]]
-We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
+We cloned UAS-TH 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 uTHtest.png|center|400px|标题]]
 [http://2017.igem.org/Team:Tongji_China/Design More Information]
@@ Line 64: / Line 73: @@
-===Design Notes===
-'''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 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]]
-We also did one site direct mutagenesis for submission:<br>
-Pst I (647) CTGCAG->CTGCTG