Difference between revisions of "Part:BBa K2374004"

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 218
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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.

@@ Line 23: / Line 23: @@
 ===Design notes===
-[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]]
-[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]]
-[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]]
 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 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].
+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>
-site direct mutagenesis for submission:
-Xba I (708): TCTAGA->TCAAGA
-Pst I (1353): CTGCAG->CTGCTG
-We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
+We cloned GAL4 into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.
 [[File:2017tongji image registry 4test.png|center|400px|标题]]
 [http://2017.igem.org/Team:Tongji_China/Design More Information]
+site direct mutagenesis for submission:<br>
+. Xba I (708): TCTAGA->TCAAGA <br>
+. Pst I (1353): CTGCAG->CTGCTG
 ===Test Results===
@@ Line 56: / Line 57: @@
 <partinfo>BBa_K2374004 SequenceAndFeatures</partinfo>
+===References===
+Christian Dahmann. ''Drosophila'' Methods and Protocols 2nd Edition. New York: Humana Press, 2016: 33-38.
 ===Functional Parameters===
 <partinfo>BBa_K2374004 parameters</partinfo>
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