Difference between revisions of "Part:BBa K2374007"
(→‎Design notes)
 
(21 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2374007 short</partinfo>
 
<partinfo>BBa_K2374007 short</partinfo>
  
−
ple (Tyrosine 3-monooxygenase, TH)
+
===Overview===
 +
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 +
! colspan="2" style="background:#FFBF00;"|UAS-TH
 +
|-
 +
|'''Use in'''
 +
|''D.melanogaster''
 +
|-
 +
|'''RFC standard'''
 +
|
 +
|-
 +
|'''Backbone'''
 +
|pSB1C3<br>
 +
|-
 +
|'''Submitted by'''
 +
|[http://2017.igem.org/Team:Tongji_China Tongji_China 2017]
 +
|}
  
−
<!-- Add more about the biology of this part here
+
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.
−
===Usage and Biology===
+
  
 +
 +
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]).
 +
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>
 +
1.Tyrosine 3-monooxygenase ('''ple''') <br>
 +
2.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===
 +
 +
'''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 synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pUAST-UAS-TH. The pUAST-UAS-TH also with the other two plasmids: pUAST-pleP-GAL4 ([https://parts.igem.org/Part:BBa_K2374005 BBa_K2374005])and pUAST-pleP-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|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>
 +
'''''note''''':
 +
1. 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>
 +
2. 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]
 +
 +
 +
 +
Here shows the restriction endonuclease digestion image of pUAST-UAS-TH.
 +
 +
[[File:2017tongji_registry_image_UTH.png|center|300px|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.<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]
 +
 +
===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>
 
<span class='h3bb'>Sequence and Features</span>
Line 13: Line 74:
  
  
−
<!-- Uncomment this to enable Functional Parameter display
+
 
 +
 
 +
 
 +
 
 
===Functional Parameters===
 
===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 [https://www.ncbi.nlm.nih.gov/gene/?term=38746]
 +
 +
FlyBase [http://flybase.org/reports/FBgn0005626.html]
 +
 +
 
<partinfo>BBa_K2374007 parameters</partinfo>
 
<partinfo>BBa_K2374007 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
===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.<br>
 +
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.<br>
 +
3. Lewandoski Mark (2001). Conditional control of gene expression in the mouse. ''Nature Reviews Genetics''. 2: 743–755.

Latest revision as of 03:50, 2 November 2017


UAS-TH -> (fruit fly)

Overview

UAS-TH
Use in D.melanogaster
RFC standard
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.

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 synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pUAST-UAS-TH. The pUAST-UAS-TH also with the other two plasmids: pUAST-pleP-GAL4 (BBa_K2374005)and pUAST-pleP-GAL80ts (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.
The result of our testing on D.melanogaster is displayed below.

pleP-GAL4


pleP-GAL80ts


pleP-GAL80ts


note: 1. 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.
2. 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.
[http://2017.igem.org/Team:Tongji_China/Design More Information]


Here shows the restriction endonuclease digestion image of pUAST-UAS-TH.

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.

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]

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
    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




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.