Part:BBa_K3734029

TetR-ELK1

TetR is a kind of protein that can find and combined with TRE DNA structure domain. ELK is a kind of protein that can activate downstream gene expression after being regulated by phosphorylation

TetR-ELK

TetR can recognize and combine with TRE, it is a important part of Tet-off system. Without tetracycline, TetR can combine with TRE. With the presence of tetracycline, the combination of TetR and TRE will be blocked and the Tet-off system will be shut down. ELK is generally dormant, when INSR receive insulin and activate MAPK, ELK will be phophated and activated, then ELK is able activate expression of target gene downstream TRE.

1.Pattern diagram

Fig.1 The model diagram TetR-ELK

2.1 Method

After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.

We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.

2.2 Result

Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system

Fig.3 ERK phosphorylation changes with different insulin treatment

Fig.4 ERK phosphorylation changes with different insulin treatment

3.Caution

Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting

Reference：

[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 750
  
• 1000
  COMPATIBLE WITH RFC[1000]