Difference between revisions of "Part:BBa R0071:Experience"
 
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__NOTOC__
 
__NOTOC__
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<br><br><br>[[Image:Improved_Prhl_Promoter_Assay_Result.png|thumb|left|430px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]]
 
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[[Image:Improved_Prhl_Promoter_Assay_Extracted_Result.png|thumb|right|440px|<b>Fig. 2. </b> The fluorescence intensity of the cells with the original Prhl, Prhl(RR), and Prhl(RL)]]
 
  
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===Improvement and Characterize===
  
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We, Tokyo Tech 2014, improved Prhl Promoter (BBa_R0071) by changing LuxR binding site of Plux Promoter to RhlR binding site.  
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Group: <b>Tokyo Tech 2016</b>
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We measured the GFP expression with the four different promoters (Prhl, Prhl(RR) (BBa_K1529320), Prhl(LR) (BBa_K1529310), and Prhl(RL)(BBa_K1529300)) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL.<br>
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Author: Yoshio Takata
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Summary of Improvement and Characterization:
 +
 
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I. Improved Prhl by iGEM 2014 Tokyo_Tech and characterize RhlR assay 
 +
 
 +
II. Improvement of the wild type Prhl
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III. Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech to our original improved Prhl.
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[[Image:prhl4.png|thumb|center|400px|Fig. 3 RFU of GFP / turbidity of imoroved Prhl mutants]]<br>
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The sequences of these two chosen mutants are shown in below.
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Native Prhl (<partinfo>BBa_R0071</partinfo>)
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TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAAAAAGTGTTCtactagagAAAGAGGAGAAA
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Prhl(D6) (<partinfo>BBa_K1949060</partinfo>)
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TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTA<b>t</b>AAAGTGTTCtactagagAAAGAGGAGAAA
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Prhl(H7) 
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TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTA<b>t</b>AAAGTGTTCtactag<b>ta</b>AAAGAGGAGAAA                 
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If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech  our work in Tokyo_Tech 2016 wiki]!
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===Improvement===
 +
 
 +
====iGEM 2014 Tokyo_Tech====
 +
 
 +
[[Image:Improved_Prhl_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]]
 +
 
 +
 
 +
We, Tokyo Tech 2014, improved Prhl Promoter (<partinfo>BBa_R0071</partinfo>) by changing LuxR binding site of Plux Promoter to RhlR binding site.<br>
 +
We dsigned new Prhl promoter
 +
We measured the GFP expression with the four different promoters (Prhl : BBa_R0071, Prhl(RR) : <partinfo>BBa_K1529320</partinfo>, Prhl(LR) :  <partinfo>BBa_K1529310</partinfo> and Prhl(RL) : <partinfo>BBa_K1529300</partinfo>) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL.<br>
 
Fig. 1 shows the fluorescence intensity detected by flow cytometer. <br>
 
Fig. 1 shows the fluorescence intensity detected by flow cytometer. <br>
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Fig. 2 is the extracted data which shows the comparison of the promoters: Prhl, Prhl(RR), and Prhl(RL).<br>
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From the result, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter (BBa_R0071).<br>  
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As Fig. 2 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter.<br>  
+
 
Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. <br>
 
Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. <br>
 
This means Prhl(RL) promoter has little leak.<br>
 
This means Prhl(RL) promoter has little leak.<br>
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Therefore, we can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.
+
Therefore, we can say that Prhl(RL):<partinfo>BBa_K1529300</partinfo> promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.
  
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<br>
 
 
<br>
 
<br>
 
For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay our work in Tokyo_Tech 2014 wiki].
 
For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay our work in Tokyo_Tech 2014 wiki].

Latest revision as of 03:37, 16 October 2016


Improvement and Characterize

Group: Tokyo Tech 2016

Author: Yoshio Takata

Summary of Improvement and Characterization:

I. Improved Prhl by iGEM 2014 Tokyo_Tech and characterize RhlR assay

II. Improvement of the wild type Prhl

III. Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech to our original improved Prhl.

Fig. 3 RFU of GFP / turbidity of imoroved Prhl mutants

The sequences of these two chosen mutants are shown in below.

Native Prhl (BBa_R0071)

TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAAAAAGTGTTCtactagagAAAGAGGAGAAA

Prhl(D6) (BBa_K1949060)

TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagagAAAGAGGAGAAA

Prhl(H7)

TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAtAAAGTGTTCtactagtaAAAGAGGAGAAA

If you want more information, you see [http://2016.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2016 wiki]!

Improvement

iGEM 2014 Tokyo_Tech

Fig. 1. Fluorescence intensity detected by flow cytometer


We, Tokyo Tech 2014, improved Prhl Promoter (BBa_R0071) by changing LuxR binding site of Plux Promoter to RhlR binding site.
We dsigned new Prhl promoter We measured the GFP expression with the four different promoters (Prhl : BBa_R0071, Prhl(RR) : BBa_K1529320, Prhl(LR) : BBa_K1529310 and Prhl(RL) : BBa_K1529300) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL.
Fig. 1 shows the fluorescence intensity detected by flow cytometer.
From the result, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter (BBa_R0071).
Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio.
This means Prhl(RL) promoter has little leak.
Therefore, we can say that Prhl(RL):BBa_K1529300 promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.


For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay our work in Tokyo_Tech 2014 wiki].

Applications of BBa_R0071

User Reviews

UNIQ9f48a09bc762e062-partinfo-00000007-QINU UNIQ9f48a09bc762e062-partinfo-00000008-QINU

No review score entered. Northwestern 2011

The 2011 Northwestern iGEM team used this promoter as a unit within our Pseudomonas Aeruginosa biosensor. When this RhlR/C4-HSL regulated promoter is induced at varying concentrations of C4-HSL, we observed GFP fluorescence in accordance to the graph below.

RhlR promoter.jpg

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