Part:BBa_K3734016

9XUAS

UAS is a DNA structure domain that combined with GAL4.

9XUAS

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_K3734023) 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.Pattern diagram

Fig.1 The model diagram of 9XUAS-Insulin

2.1 Method

We use LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells.

2.2 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.2 Light controlled system testing experiment

Then we changed the downstream target gene into insulin gene and proved that the insulin can be expressed, processed and secreted successfully and the efficiency of 9XUAS is high enough.

Fig.3: Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively

3.Caution

Because we have repeated 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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]