Difference between revisions of "Part:BBa K2374004"

Revision as of 21:02, 30 October 2017

GAL4 (galactose-responsive transcription factor)

Overview

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_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].

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

[http://2017.igem.org/Team:Tongji_China/Design More Information]

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]

Sequence and Features