Difference between revisions of "Part:BBa K2036027"
 
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This is the whole characterization circuit of Signal Filter prokaryote version(http://2016.igem.org/Team:HUST-China) as Fig1 shows.
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This is the whole characterization circuit of Signal Filter prokaryote version([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki ]) as Fig1 shows.
 
[[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>Here is how we conduct our characterization: We employ T7 and ptrp as our input sensors and BL21 as host strain. When induced by IPTG,Cro and CII are expressed under T7 promoter and CII will give a positive feedback to enhance Cro's expression by promoting pRE. Accumulated Cro will stably bind to the bingding site within pRM thus repress GFP and at the same time RFP expression will not be interrupted (see to Fig2)   
 
<br>Here is how we conduct our characterization: We employ T7 and ptrp as our input sensors and BL21 as host strain. When induced by IPTG,Cro and CII are expressed under T7 promoter and CII will give a positive feedback to enhance Cro's expression by promoting pRE. Accumulated Cro will stably bind to the bingding site within pRM thus repress GFP and at the same time RFP expression will not be interrupted (see to Fig2)   
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<br>When IAA is added, CI will be expressed to further block pR. And with CII's degradation by Ftsh, GFP expression will gradually comes up to a stable state. Meanwhile, RFP level will decrease by protease degradation system through LVAssrA tag. (see to Fig3)
 
<br>When IAA is added, CI will be expressed to further block pR. And with CII's degradation by Ftsh, GFP expression will gradually comes up to a stable state. Meanwhile, RFP level will decrease by protease degradation system through LVAssrA tag. (see to Fig3)
 
[[File:T--HUST-China--Experiments-Fig14-3.png|thumb|500px|center|Fig3:Prokaryote version of Signal Filter characterization plasmid]]
 
[[File:T--HUST-China--Experiments-Fig14-3.png|thumb|500px|center|Fig3:Prokaryote version of Signal Filter characterization plasmid]]
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<h3> Protein&protein reaction</h3>
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<p>
 +
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|thumb|800px|center|Fig: FtsH degradation test of CIII]]
 +
<p>
 +
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 tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.
 +
</p>
 +
<br>
 +
<h3> Protein&promoter</h3>
 +
<p>
 +
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-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.
 +
</p>
 +
[[File: T--HUST-China--CII-pRE_plate.png |thumb|800px|center|Fig: CII and pRE activation test]]
 +
<p>
 +
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.
 +
</p>
 +
[[File: T--HUST-China--Experiments-CII-pRE_Flou-detec.png|thumb|800px|center|Fig: Fluorescence detection]]
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<br>
 +
<p>
 +
CI is a repressor from bacteriophage lambda. To test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFPLVAssrAtag-PET-Duet-1 and take pR-RBS-GFPLVAssrAtag-PET-Duet-1 as control to test its inhibition function.
 +
As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.
 +
</p>
 +
[[File:T--HUST-China--Experiments-CI-pR_plate.png|thumb|800px|center|Fig: CI-pR inhibition test]]
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<p>
 +
We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below). From figure we can find the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.
 +
</p>
 +
[[File:T--HUST-China--Experiments-CI-pR_Flou-detec.png|thumb|800px|center|Fig: Fluorescence detection]]
 +
<br>
 +
<p>
 +
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.
 +
</p>
 +
[[File: T--HUST-China--CI-pR_inhibition.png |thumb|800px|center|Fig: Cro and pRM inhibition test]]
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<br>
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<h3> Tri-stable function</h3>
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<p>
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Ptrp2 (BBa_K1592024) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.
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</p>
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[[File: T--HUST-China--ptrp-IAA.png |thumb|800px|center|According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.]]
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<br>
 +
<p>
 +
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.
 +
</p>
 +
<br>
 +
[[File:T--HUST-China--Experiments-LVAssrA.png|thumb|800px|center|Fig: LVAssrAtag degradation rate measurement under plac]]
 +
<p>
 +
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.
 +
</p>
 +
<br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:15, 19 October 2016


pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag


This is the whole characterization circuit of Signal Filter prokaryote version([http://2016.igem.org/Team:HUST-China Details see to HUST-China 2016 wiki ]) as Fig1 shows.

Fig1:Prokaryote version of Signal Filter characterization circuit


Here is how we conduct our characterization: We employ T7 and ptrp as our input sensors and BL21 as host strain. When induced by IPTG,Cro and CII are expressed under T7 promoter and CII will give a positive feedback to enhance Cro's expression by promoting pRE. Accumulated Cro will stably bind to the bingding site within pRM thus repress GFP and at the same time RFP expression will not be interrupted (see to Fig2)

Fig2:Prokaryote version of Signal Filter characterization plasmid


When IAA is added, CI will be expressed to further block pR. And with CII's degradation by Ftsh, GFP expression will gradually comes up to a stable state. Meanwhile, RFP level will decrease by protease degradation system through LVAssrA tag. (see to Fig3)

Fig3:Prokaryote version of Signal Filter characterization plasmid

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.


Fig: FtsH degradation test of CIII

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 tandomly expressed CIII can efficiently protect CII from being degraded by Ftsh.


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-GFPLVAssrAtag as CK to see if CII efficiently activate pRE.

Fig: 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.

Fig: Fluorescence detection


CI is a repressor from bacteriophage lambda. To test its interaction with pR promoter, we constructed CI-TT-pR-RBS-GFPLVAssrAtag-PET-Duet-1 and take pR-RBS-GFPLVAssrAtag-PET-Duet-1 as control to test its inhibition function. As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit.

Fig: CI-pR inhibition test

We also detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification (seen from the figure below). From figure we can find the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI to pR from a more intuitive way.

Fig: Fluorescence detection


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.

Fig: Cro and pRM inhibition test


Tri-stable function

Ptrp2 (BBa_K1592024) is an improved part from HUST-China 2015, we employed it as one of our signal sensor to test our tri-stable switch. We constructed ptrp2-GFP-pSB1C3 to determine an appropriate induction concentration.

According to the GFP expression curve, IAA induction of ptrp2 with 50μM final concentration is a better choice relatively.


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.


Fig: 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
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 717
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 717
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 717
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 717
    Illegal AgeI site found at 2533
    Illegal AgeI site found at 2645
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3561