Difference between revisions of "Part:BBa K2036015"
 
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<partinfo>BBa_K2036015 short</partinfo>
 
<partinfo>BBa_K2036015 short</partinfo>
 
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CII acts as a necessary and sufficient cis-acting target for rapid proteolysis by initiating pRE. The circuit is built to test CII and pRE interaction together with RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag( [https://parts.igem.org/Part:BBa_K2036013 BBa_K2036013])and pRE-RBS-GFP-LVAssrAtag( [https://parts.igem.org/Part:BBa_K2036011 BBa_K2036011]) . Considering Ftsh protease degrades it raidly in the host, RBS-CII-RBS-CII tandom expression circuit is constructed.
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CII acts as a necessary and sufficient cis-acting target for rapid proteolysis by initiating pRE. The circuit is built to test CII and pRE interaction together with RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag( [https://parts.igem.org/Part:BBa_K2036013 BBa_K2036013])and pRE-RBS-GFP-LVAssrAtag( [https://parts.igem.org/Part:BBa_K2036011 BBa_K2036011]) . Considering Ftsh protease degrades it raidly in the host, RBS-CII-RBS-CII tandem expression circuit is constructed.
  
 
[[File:T--HUST-China--Experiments-Fig10.png |thumb|500px|center|Fig1:CII and pRE interaction characterization circuits]]
 
[[File:T--HUST-China--Experiments-Fig10.png |thumb|500px|center|Fig1:CII and pRE interaction characterization circuits]]
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<partinfo>BBa_K2036015 parameters</partinfo>
 
<partinfo>BBa_K2036015 parameters</partinfo>
 
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<h2>Protein&promoter</h2>
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<p>--CII and pRE</p>
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<br>
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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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<br>
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[[File:T--HUST-China--CII-pRE_plate.png|800px|thumb|center|Fig2: 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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<br>
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[[File:T--HUST-China--Experiments-CII-pRE_Flou-detec.png|800px|thumb|center|Fig3: 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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<h2>Protein&protein reaction</h2>
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<p>
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We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.
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</p>
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<br>
 +
[[File:T--HUST-China--CIII%26Ftsh.png|800px|thumb|center|Fig4: According to the flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandemly expressed CIII can efficiently protect CII from being degraded by Ftsh. ]]
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<h2>Preliminary experiments of LVAssrA-tag</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 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|800px|thumb|center|Fig5: 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.]]

Latest revision as of 06:45, 25 October 2016


RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag
CII acts as a necessary and sufficient cis-acting target for rapid proteolysis by initiating pRE. The circuit is built to test CII and pRE interaction together with RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag( BBa_K2036013)and pRE-RBS-GFP-LVAssrAtag( BBa_K2036011) . Considering Ftsh protease degrades it raidly in the host, RBS-CII-RBS-CII tandem expression circuit is constructed.

Fig1:CII and pRE interaction characterization circuits

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 1770


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 if CII efficiently activate pRE.


Fig2: According to the flourescence 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 below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter.

Protein&protein reaction

We had submitted and documented RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036014) and RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag (BBa_K2036015). These two parts were to test whether CIII can protect CII from being degraded by Ftsh by competitive inhibition.


Fig4: According to the flourescence measurement curve above, we can see clearly that GFP level of CIII test circuit increased over time and it showed significant difference from two control groups. It indicates that tandemly expressed CIII can efficiently protect CII from being degraded by Ftsh.

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.


Fig5: 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.