Difference between revisions of "Part:BBa K2374002:Design"

(→‎Design Notes)
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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We use PCR to clone the GAL80ts from the genomic DNA of ''Saccharomyces cerevisia'' S288C.
 
  
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Then to make 2 site-directed mutagenesis:
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We use PCR to clone the GAL80ts from the genomic D
 +
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].
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[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]  <br> <br>
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[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]  <br><br>
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[[File:2017tongji_image_registry_uTH.png|center|200px|pleP-GAL80ts]]  <br><br>
  
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EcoR I (325):GAATTC->GAGTTC
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There are other mutations during the parts construction by accident:
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We cloned GAL80ts into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
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1142: A->G; aa: Lys->Arg
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[[File:2017tongji image registry 80test.png|center|400px|标题]]
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1154: A->G; aa: Glu->Gly
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We did 2 mutagenesis on this sequence.<br>
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site direct mutagenesis:<br>
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1. EcoR I (184)  GAATTC->GATTTC <br>
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2. Xba I  (219)  TCTAGA->TGTAGA
  
 
===Source===
 
===Source===

Revision as of 20:33, 30 October 2017


GAL80ts (temperature dependent)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 993
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 19
    Illegal BglII site found at 615
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 27
    Illegal BsaI site found at 73


Design Notes

We use PCR to clone the GAL80ts from the genomic D 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_K2374003 [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].

pleP-GAL4


pleP-GAL80ts


pleP-GAL80ts



We cloned GAL80ts 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

Chromosome: XIII; NC_001145.3 (171594..172901) NOTE: Gal80ts is a mutation based on the gene above.


References