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.
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.
And the data of cytokine secretion, shows that the secretion get to its highest peak around 40 hours.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 120
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 167
Illegal XhoI site found at 49 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 189
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1581
Illegal SapI.rc site found at 210
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