Composite

Part:BBa_K2036011

Designed by: Zhangyu Cheng   Group: iGEM16_HUST-China   (2016-09-19)
Revision as of 05:52, 25 October 2016 by ZhiZeng (Talk | contribs)

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pRE-GFP-LVAssrAtag
It is a GFP generator,and the production of GFP will be activated by a certain level of CII or CII with help of CIII in E.coli. HUST-China 2016 built this circuit to characterize CII and pRE interaction with test group: RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036013) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015).

Fig1:CII&pRE interaction characterization circuit

Protein&promoter

--CII and pRE


CII (BBa_K2036000) functions as a transcriptional activator to direct promoter RE, so we constructed CII-TT-pRE-RBS-GFP-LVAssrAtag as test group and pRE-RBS-GFP-LVAssrAtag as CK to see whether CII can efficiently activate pRE.


Fig2: According to the Fluorescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.


Fig3: We also did Fluorescence microscope detection after 30, 120 and 240 minutes' induction. According to the figture above, we can tell qualitatively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

Preliminary experiments of LVAssrAtag

In order to prove that our toolkit is efficient to switch two interest genes’ expression from GFP to RFP and to eliminate the accumulation of expressed protein to interfere our measurement. We fused a degradation tag at the amino terminal of our reporter. And we used plac from the Registry(BBa_J04500) to characterize the degradation tag LVAssrA. We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with excitation wavelength 495nm.


Fig4: From the figure above, we are sorry to find that plac can not be prohibited from leakage, as there are nearly no difference between the test and control group. But we are confident to prove the high degradation efficiency of the tag as more than two thirds of the GFP degraded within 90 minutes which also offered an interesting and useful tool for rapidly down regulating certain target protein.


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 BsaI.rc site found at 719


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