Difference between revisions of "Part:BBa K2374001:Design"

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===Design Notes===
 
===Design Notes===
  
−
We get this 452bp sequence from FlyBase ( Dmel\ple [http://flybase.org/reports/FBgn0005626.html]). We regard the 5'-UTR region as promoter region of ''ple''.
+
We get this 452bp sequence from FlyBase ( Dmel\ple [http://flybase.org/reports/FBgn0005626.html]). We regard the 5'-UTR region as promoter region of '''ple'''.<br>
−
This should not be the most accurate and concise promoter sequence. We hope that subsequent users will be able to accurately identify the promoter sequence.
+
This should not be the most accurate and concise promoter sequence. We hope that subsequent users will be able to accurately identify the promoter sequence.<br><br>
−
We cloned this 452bp TH promoter easily from ''D. melanogaster'' 's cDNA library and the sequencing result is correct.
+
−
We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells.
+
−
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] )
+
  
 +
We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells. (More details [http://2017.igem.org/Team:Tongji_China/Design])<br>
 +
We cloned this 452bp TH promoter easily from ''D. melanogaster'' '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. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.
−
site direct mutangenesis:
+
[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br> <br> <br>
−
EcoR I (184)  GAATTC->GATTTC
+
[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br>
−
Xba I  (219)  TCTAGA->TGTAGA
+
We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
 +
[[File:2017tongji image registry ptest.png|center|400px|标题]]
 +
We did 2 mutagenesis on this sequence.<br>
 +
site direct mutagenesis:<br>
 +
1. EcoR I (184)  GAATTC->GATTTC <br>
 +
2. Xba I  (219)  TCTAGA->TGTAGA
  
 
===Source===
 
===Source===
  
−
NT_037436.4 (NCBI)
+
GeneCards®: The Human Gene Database
 +
 
 +
NCBI
 +
 
 +
FlyBase
  
 
===References===
 
===References===
 +
 +
Harrington CA, Lewis EJ, Krzemien D, Chikaraishi DM. Identification and cell type specificity of the tyrosine hydroxylase gene promoter. ''Nucleic Acids Research''. 1987;15(5):2363-2384.

Latest revision as of 02:58, 2 November 2017


TH (ple) promoter-> (fruit fly)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 137
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We get this 452bp sequence from FlyBase ( Dmel\ple [http://flybase.org/reports/FBgn0005626.html]). We regard the 5'-UTR region as promoter region of ple.
This should not be the most accurate and concise promoter sequence. We hope that subsequent users will be able to accurately identify the promoter sequence.

We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells. (More details [http://2017.igem.org/Team:Tongji_China/Design])
We cloned this 452bp TH promoter easily from D. melanogaster 's genomic DNA and the sequencing result is correct.

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. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.

pleP-GAL4



pleP-GAL80ts

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

标题

We did 2 mutagenesis on this sequence.
site direct mutagenesis:
1. EcoR I (184) GAATTC->GATTTC
2. Xba I (219) TCTAGA->TGTAGA

Source

GeneCards®: The Human Gene Database

NCBI

FlyBase

References

Harrington CA, Lewis EJ, Krzemien D, Chikaraishi DM. Identification and cell type specificity of the tyrosine hydroxylase gene promoter. Nucleic Acids Research. 1987;15(5):2363-2384.