Composite

Part:BBa_K2986008

Designed by: zheng shuxin   Group: iGEM19_SUSTech_Shenzhen   (2019-10-16)
Revision as of 08:23, 19 October 2019 by Susan1999 (Talk | contribs)


5*UAS-mRuby-hGluc-IL10

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. we added the target gene Interleukin 10(IL-10), we wanted to use hGluc and mRuby to indicate the expression and secretion process of IL-10.

Figure1.the plasmid contain 5xUAS-mRuby-hGluc-IL10

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-10 expression with the control group. The data showed that inside Hela cell, the mRuby indicator system has little influence on cytokine expression and secretion. This showed that the Characterization of system was reliable.

Figure2.cell(with IL10 expression) number with light exposure
Figure3.cell number with light exposure of negative control group and experiment group


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 BglII site found at 1874
    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


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