Difference between revisions of "Part:BBa K2036009"
 
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<partinfo>BBa_K2036009 short</partinfo>
 
<partinfo>BBa_K2036009 short</partinfo>
  
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pRM-GFP-LVAtag, the circuit can function as control group of Cro and pRM interaction characterization with test group: Cro-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K20360010 BBa_K20360010]).
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pRM-GFP-LVAssrAtag, the circuit can function as a control group of Cro and pRM interaction characterization with test group: Cro-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K20360010 BBa_K20360010]).
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<br>When inserted into expression plasmid and tramsformed into E.coli. Take PETDuet-1 as an example, IPTG can trigger T7 promoter in the plasmid, and  cro can bind to a certain site within pRM, so that GFP will not be produced. And we get a NO gate of GFP generator.
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[[File:T--HUST-China--Experiments-Fig12.png|thumb|400px|center|Fig1:Cro&pRM interaction characterization circuit]]
 
[[File:T--HUST-China--Experiments-Fig12.png|thumb|400px|center|Fig1:Cro&pRM interaction characterization circuit]]
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<h2>Protein&promoter</h2>
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<p>--Cro and pRM</p>
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<br>
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[[File:T--HUST-China--CI-pR_inhibition.png|800px|thumb|center|Fig2: We characterized Cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains Cro expressed less GFP protein than control group over time. It proves that Cro can effectively bind pRM to block its downstream gene’s transcription.]]
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<br>
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<h2>Preliminary experiments of LVAssrAtag</h2>
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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 Registry([https://parts.igem.org/Part:BBa_J04500 BBa_J04500])  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 wavelength 495nm.
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</p>
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<br>
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[[File:T--HUST-China--Experiments-LVAssrA.png|800px|thumb|center|Fig3: 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.]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 05:46, 25 October 2016


pRM-GFP-LVAssrAtag

pRM-GFP-LVAssrAtag, the circuit can function as a control group of Cro and pRM interaction characterization with test group: Cro-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K20360010).

Fig1:Cro&pRM interaction characterization circuit

Protein&promoter

--Cro and pRM


Fig2: We characterized Cro and pRM inhibition by the same method as CI and pR’s. From line chart and fluorescence detection, we can see that the test group contains Cro expressed less GFP protein than control group over time. It proves that Cro can effectively bind pRM to block its downstream gene’s transcription.


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.


Fig3: 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 699