Difference between revisions of "Part:BBa K2374001:Design"
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We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells. <br> | We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells. <br> | ||
| − | We cloned this 452bp TH promoter easily from ''D. melanogaster'' 's | + | 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. | 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. | ||
| + | [[File:2017tongji_image_registry_ple4.png|left|200px|标题]] | ||
| + | [[File:2017tongji_image_registry_ple80.png|right|200px|标题]] | ||
We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image. | We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image. | ||
Revision as of 07:53, 30 October 2017
TH (ple) promoter-> (fruit fly)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 137
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
We did 2 mutagenesis on this sequence.
site direct mutagenesis:
1. EcoR I (184) GAATTC->GATTTC
2. Xba I (219) TCTAGA->TGTAGA
Source
NT_037436.4 (NCBI)