Coding

Part:BBa_K2200007

Designed by: Mixiao Gui   Group: iGEM17_Shenzhen_SFLS   (2017-09-29)
Revision as of 16:17, 30 October 2017 by Awesome Zz (Talk | contribs)


Gal4 transcription factor is a positive regulator.

GAL4 is a transcription factor of galactose metabolism related genes, which recognizes and binds the promoter UAS through its N-terminal DNA binding domain, and assembles and transcribes the RNA polymerase II complex through its C-terminal activation domain.According to previous studies, it's activity can be up-regulated by adding the P65 sequence. 

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Usage and Biology

The function of this part was demonstrated by an experiment in a previous paper. CMV promotor was selectively inserted in GAL4-P65,GAL4 and GAL4-VP64 gene.(VP64 was a transcription factor) And GAL4-P65 was also inserted downsream the tumor-specific promotor hTERT. The function of these promotors and transcription factors were measured by the expression of luciferase in bladder cancer cell lines T24 , 5637, and human human foreskin fibroblasts. The results showed that after adding the sequence of P65, the luciferase activities were obviously up-regulated by the CMV-GAL4-P65 vector group, which means that the GAL4P65 system can significently enhance the expression of downstream gene comparing with the GAL4.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 710
    Illegal XhoI site found at 218
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 886
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 137


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Functional Parameters

The function of this TAS part was demonstrated by an experiment in a previous paper [1]. CMV promotor was selectively inserted in GAL4-P65, GAL4 and GAL4-VP64 (VP64, a transcription factor) gene. GAL4P65 was also inserted downstream the tumor-specific promotor hTERT. The function of these promotors and transcription factors were measured by the expression of fluoresce in bladder cancer cell lines T24, 5637, and human foreskin fibroblasts.

The results in the paper ([1]-fig.2) showed that the luciferase activities were obviously up-regulated by the CMV-GAL4-P65 vector group, which means it can enhance the expression of downstream gene.

Reference

[1] Huang X, Zhuang C, Zhuang C, et al. An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells. Molecular Biosystems, 2017, 13(9): 1713-1721.

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