Part:BBa_K2986009

5*UAS-mRuby-hGluc-IL8

Usage and Biology

We construct a plasmid that can be controlled by blue light, it was composite with the 5xUAS-mRuby-hGluc-IL8. We used a light-switch transactivator named GVAPO, which can bind to promoter and initiates transcription of upstream gene in a short time. And 5xUASis a site for GVAPO binding and perform its function. After light activation, GVAP homodimerizes and interacts with this sequence to initiates expression of gene interest.We use mRuby to be a reporter of our target gene expression (cytokine secretion), and to obtain better indicators for long-term observation, we need to replace cytokines with Humanized Gaussia luciferase (hGluc). P2A is 2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains. we added the target gene Interleukin 8(IL-8), we wanted to use hGluc and mRuby to indicate the expression and secretion process of IL-8.

Figure1.plasmid with 5xUAS-mRuby-hGluc-IL8

Properties

We used hGluc as a long-term indicator for the target gene expression, and we get the data after 48 hrs blue light exposure. We contrasted IL-8 expression with the control group. we can see from the data that IL-8 transcription process is influenced by the light exposure time.

Figure2.IL-8 mRNA expression amount under blue light exposure

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 120
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 167
  Illegal BamHI site found at 906
  Illegal XhoI site found at 49
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 189
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1527
  Illegal SapI.rc site found at 210