Part:BBa_K2986012

5*UAS-mRuby-P2A-hGluc

Usage and Biology

We construct a plasmid that can be controlled by blue light, it was composite with the 5xUAS-mRuby- P2A-hGluc. 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.

Figure1.the plasmid used with hgluc

Properties

We used hGluc as a long-term indicator for the target gene expression, and we get the data after 70 hrs blue light exposure. We found that the expression amount of cytokines reach to a peak within 20 hours, then go down, reach the second between 20 to 40 hours, and the third peak is about 60hours.

Figure2.cell number after blue light exposure test through hGluc

And the data of cytokine secretion, shows that the secretion get to its highest peak around 40 hours.

Figure3.hGluc activity after blue light exposure

Sequence and Features

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 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 1581
  Illegal SapI.rc site found at 210