Composite

Part:BBa_K3734032

Designed by: Xingjun Zhao   Group: iGEM21_CSU_CHINA   (2021-10-11)
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9XUAS-Ad-LUC

UAS is a DNA structure domain that combined with GAL4. Luciferin (LUC) is a protein that can activate fluorescein.We use it as reporter gene.

9XUAS-Ad-LUC

UAS (BBa_K758003)) is a piece of small DNA sequence that can be recognized and combined by GAL4 DBD. Team iGEM12_KIT-Kyoto repeated UAS 5 times when using it. In our design, considering of that the mutual effect of our blue light protein would reduce the efficiency and the whole pathway is relatively long, while the react time of our system should be as short as possible, we have made some alterations

Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin(BBa_K3734033) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.Firstly, we have repeated the UAS before promoter for 9 times. The purpose of constructing 9XUAS-Ad-LUC (BBa_K3734032) and 9XUAS-Ad-insulin( BBa_K3734033) is to enhance the recognition and combination of GAL4 DBD, improve the efficiency of downstream target gene activation.

Secondly, we have used Ad promoter as promoter to make it able to function in eukaryotic cells and improve the expression efficiency after the activation of VP16

1.Experiment

1.1 Method

We used report gene LUC to represent the working status of 9XUAS-Ad

1.2 Result

we used the reporting gene LUC as the downstream target gene, and set up a positive control group and a negative control group. The results prove that the efficiency of our improved 9XUAS has been greatly improved, which can meet the requirements of short loop response

Fig.1 Light controlled system testing experiment

2.Caution

Because we repeat UAS nine times, we should pay attention to avoiding duplicate fragments when designing primer. If you can't avoid duplicate clips, please note the times that UAS in PCR products has been repeated.

Reference：

[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.

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 38
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 301
    Illegal NgoMIV site found at 1645
    Illegal NgoMIV site found at 1666
    Illegal AgeI site found at 1369
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1551


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