Difference between revisions of "Part:BBa K2036019"
 
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<partinfo>BBa_K2036019 short</partinfo>
 
<partinfo>BBa_K2036019 short</partinfo>
  
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This is a part of Prokaryote version of Signal Filter ([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LVAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036027 BBa_K2036027]).
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This is a part of Prokaryote version of Signal Filter ([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag ([https://parts.igem.org/Part:BBa_K2036027 BBa_K2036027]).
 
[[File:T--HUST-China--Experiments-Fig14-1.png|thumb|500px|center|Fig1:Prokaryote version of Signal Filter characterization circuit]]
 
[[File:T--HUST-China--Experiments-Fig14-1.png|thumb|500px|center|Fig1:Prokaryote version of Signal Filter characterization circuit]]
 
<br>Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.  
 
<br>Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.  
 
[[File:T--HUST-China--Experiments-Fig13.png|thumb|350px|center|Fig2:Prokaryote version of Signal Filter characterization plasmid]]
 
[[File:T--HUST-China--Experiments-Fig13.png|thumb|350px|center|Fig2:Prokaryote version of Signal Filter characterization plasmid]]
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<p>
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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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</p>
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<br>
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[[File:T--HUST-China--CI-pR_inhibition.png|thumb|800px|center|Fig3: Cro and pRM inhibition test]]
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<h3>Preliminary experiments of LVAssrA-tag</h3>
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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]) 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>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 06:57, 25 October 2016


RFP-LAAtag-TT-pRM-RBS-GFP-LVAssrAtag

This is a part of Prokaryote version of Signal Filter ([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki]) characterization circuit:pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag (BBa_K2036027).

Fig1:Prokaryote version of Signal Filter characterization circuit


Because the circuit is too complex, we divided it into three parts and each is assembled by In-Fusion method (homologous recombination). The whole circuit is built by 3A assembly and then we transfer the backbone from pSB1C3 to PET-Duet-1.

Fig2:Prokaryote version of Signal Filter characterization plasmid

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.


Fig3: Cro and pRM inhibition test

Preliminary experiments of LVAssrA-tag

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 573
    Illegal AgeI site found at 685
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1601