Part:BBa_K4516005

luciferase

luciferase

Contribution

The Luciferase reporter system is a reporter system which uses luciferin as substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation process, luciferin will emit bioluminescence. The bioluminescence released during oxidation of luciferin can then be measured by a fluorescence detector, also known as a luminometer or a liquid flash detector. The bioluminescence system, luciferin and luciferase, can detect gene expression very sensitively and efficiently. It is a method to detect the interaction between transcription factors and DNA in the promoter region of target gene.

Transcription factor is a kind of protein molecule with special structure and function of regulating gene expression, also known as trans-acting factor. Some transcription factors bind only to specific sequences in their target promoters. These specific sequences are called cis-acting elements. The DNA binding domain of transcription factors and cis-acting elements achieve covalent binding, thus inhibiting or enhancing gene expression. Luciferase reporter assay (luciferase assay) is an important method to detect the specific sequence binding between these transcription factors and their target promoters

Engineering

To confirm the activity of hFoxO1 protein to activate downstream gene expression, we chose two kinds of luciferase for detection. What’s more, we also chose AS1842856, which is a small component that can inhibit the activity of hFoxO1, as control group.

Fig.1hFoxO1 activity verification through luciferase activity detection..

As shown in Fig.1, after the addition of different concentrations of positive control (AS1842856), we could see a gradient of luciferin activity and concentration dependence, proving that positive control has an inhibitory effect on hFoxO1 transcriptional activation. This means the hFoxO1 transcription activation platform constructed by us is successful and can be used for follow-up experiments.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 808