Difference between revisions of "Part:BBa K2036032"
 
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<partinfo>BBa_K2036032 short</partinfo>
 
<partinfo>BBa_K2036032 short</partinfo>
  
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CII-TT-pRE-GFP-LVAssrAtag<br>
 
 
Genetic circuit for verifying the function of pRE promoter under the enhancing of CII.<br>
 
Genetic circuit for verifying the function of pRE promoter under the enhancing of CII.<br>
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Main function:<br>
 
 
We construct this circuit to verify the function of pRE promoter under the enhancing of CII. And this circuit also can be the control group when we explore the enhanced effect of multi-CII toward pRE promoter.<br>
 
We construct this circuit to verify the function of pRE promoter under the enhancing of CII. And this circuit also can be the control group when we explore the enhanced effect of multi-CII toward pRE promoter.<br>
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[[File:T--HUST-China--Experiments-Fig7-1.png|thumb|500px|center|Character rating of CII-TT]]
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[[File:T--HUST-China--Experiments-Fig7-1.png|thumb|350px|center|Fig1:Characterization plasmid of CII and pRE interaction]]
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<br>
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<h3> Protein&promoter</h3>
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<p>
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CII (  [https://parts.igem.org/Part:BBa_K2036000 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 if CII efficiently activate pRE.
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</p>
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[[File: T--HUST-China--CII-pRE_plate.png |thumb|800px|center|Fig2: CII and pRE activation test]]
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<p>
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According to the flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK.
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We also did fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.
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</p>
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[[File: T--HUST-China--Experiments-CII-pRE_Flou-detec.png|thumb|800px|center|Fig3: Fluorescence detection]]
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<br>
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<p>
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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 Rgistery ([https://parts.igem.org/Part:BBa_J04500 BBa_J04500]
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) to characterize the degradation tag LVAssrA.
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We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm.
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</p>
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<br>
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[[File:T--HUST-China--Experiments-LVAssrA.png|thumb|800px|center|Fig4: LVAssrAtag degradation rate measurement under plac]]
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<p>
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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.
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</p>
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<br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 06:55, 25 October 2016


CII-TT-pRE-RBS-GFP-LVAssrAtag

Genetic circuit for verifying the function of pRE promoter under the enhancing of CII.
We construct this circuit to verify the function of pRE promoter under the enhancing of CII. And this circuit also can be the control group when we explore the enhanced effect of multi-CII toward pRE promoter.

Fig1:Characterization plasmid of CII and pRE interaction


Protein&promoter

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 if CII efficiently activate pRE.

Fig2: CII and pRE activation test

According to the flourescence measurement curve above, we can see clearly that GFP level increased over time and it showed significant difference from CK. We also did fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

Fig3: Fluorescence detection


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 Rgistery (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 light 495nm.


Fig4: LVAssrAtag degradation rate measurement under plac

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 1142