Difference between revisions of "Part:BBa K2374004"
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===Design notes=== | ===Design notes=== | ||
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GAL4 is a important part of UAS/GAL4 system to achieve gene expression in specific cells. <br> | GAL4 is a important part of UAS/GAL4 system to achieve gene expression in specific cells. <br> | ||
We cloned this 2646bp GAL4 sequence easily from ''D. melanogaster'' (Gifts by Chunyang Ni)'s genomic DNA and the sequencing result is correct. | We cloned this 2646bp GAL4 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 ( | + | 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_K2374007 [https://parts.igem.org/Part:BBa_K2374007]). 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]. |
| + | [[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> | ||
| − | + | We cloned GAL4 into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image. | |
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| − | We cloned | + | |
[[File:2017tongji image registry 4test.png|center|400px|标题]] | [[File:2017tongji image registry 4test.png|center|400px|标题]] | ||
[http://2017.igem.org/Team:Tongji_China/Design More Information] | [http://2017.igem.org/Team:Tongji_China/Design More Information] | ||
| + | site direct mutagenesis for submission:<br> | ||
| + | 1. Xba I (708): TCTAGA->TCAAGA <br> | ||
| + | 2. Pst I (1353): CTGCAG->CTGCTG | ||
===Test Results=== | ===Test Results=== | ||
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[http://2017.igem.org/Team:Tongji_China/Experiments More details] | [http://2017.igem.org/Team:Tongji_China/Experiments More details] | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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<!-- --> | <!-- --> | ||
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<partinfo>BBa_K2374004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2374004 SequenceAndFeatures</partinfo> | ||
| − | + | ===References=== | |
| − | + | Christian Dahmann. ''Drosophila'' Methods and Protocols 2nd Edition. New York: Humana Press, 2016: 33-38. | |
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K2374004 parameters</partinfo> | <partinfo>BBa_K2374004 parameters</partinfo> | ||
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Latest revision as of 03:14, 2 November 2017
GAL4 (galactose-responsive transcription factor)
Overview
| GAL4 | |
|---|---|
| Use in | D.melanogaster |
| RFC standard | RFC 10 compatible |
| Backbone | pSB1C3 |
| Submitted by | [http://2017.igem.org/Team:Tongji_China Tongji_China 2017] |
In yeast, GAL4 regulates the galactose metabolism . Transcriptional activation of galactose utilization genes occurs when GAL4 binds to the upstream activation sequence (UAS) containing varying numbers of a 17-mer repeat. GAL4 binds to DNA as a dimer through a Zn(2)-Cys(6) zinc finger. The N-terminal region mediates both dimerization and DNA binding and contains a nuclear localization signal, while an acidic C-terminal domain controls transcriptional activation. GAL4 directly interacts with the Tra1 component of the SAGA complex, recruiting Mediator and the general transcriptional machinery to initiate transcription. This ability to activate transcription is retained when GAL4 is expressed in other species including plants, human cell lines, zebrafish, and Drosophila.
Design notes
GAL4 is a important part of UAS/GAL4 system to achieve gene expression in specific cells.
We cloned this 2646bp GAL4 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 [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_K2374007 [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].
We cloned GAL4 into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
[http://2017.igem.org/Team:Tongji_China/Design More Information]
site direct mutagenesis for submission:
1. Xba I (708): TCTAGA->TCAAGA
2. Pst I (1353): CTGCAG->CTGCTG
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 218
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 137
Illegal BsaI.rc site found at 1813
Illegal SapI site found at 502
References
Christian Dahmann. Drosophila Methods and Protocols 2nd Edition. New York: Humana Press, 2016: 33-38.