Difference between revisions of "Part:BBa K1529301"
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Prhl(RL) is a promoter that is activated by C4HSL.  <br>
 
Prhl(RL) is a promoter that is activated by C4HSL.  <br>
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We improved Prhl (<partinfo>BBa_R0071</partinfo>)  by changing LuxR binding site of Plux (<partinfo>BBa_R0062</partinfo>)  to RhlR binding site. (Fig.1)<br>  
+
We improved Prhl (<partinfo>BBa_R0071</partinfo>)  by changing LuxR binding site of Plux (<partinfo>BBa_R0062</partinfo>)  to RhlR binding site. (Fig.1 .)<br>  
−
The bottom structures of LuxR and RhlR are similar to each other, so RhlR can also bind to Lux Box. (Fig.2)  <br>
+
The bottom structures of LuxR and RhlR are similar to each other, so RhlR can also bind to Lux Box. (Fig. 2.)  <br>
  
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On the downstream of the promoter, <i>GFP</i>(<partinfo>BBa_J54103</partinfo>) is inserted as a reporter.<br>  
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On the downstream of the promoter, GFP(<partinfo>BBa_J54103</partinfo>) is inserted as a reporter.<br>  
  
  
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[[Image:titech2014_parts_luxR_and_rhlR_difference_main_Fig2.png|thumb|center|550px|<b>Fig. 2.</b> Difference between LuxR and RhlR]]
 
[[Image:titech2014_parts_luxR_and_rhlR_difference_main_Fig2.png|thumb|center|550px|<b>Fig. 2.</b> Difference between LuxR and RhlR]]
  
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To characterize the function of the <i>rhl(RL)</i> promoter (<partinfo>BBa_K1529300</partinfo>), we constructed this part Prhl(RL)_GFP (<partinfo>BBa_K1529301</partinfo>) by inserting ''rhl(RL)'' promoter upstream of a GFP coding sequence. <br
+
To characterize the function of the Prhl(RL) promoter (<partinfo>BBa_K1529300</partinfo>), we constructed this part, Prhl(RL)_GFP (<partinfo>BBa_K1529301</partinfo>), by inserting Prhl(RL) promoter upstream of a GFP coding sequence. <br
 
>By using reporter cells that contain Prhl(RL)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL. <br>  
 
>By using reporter cells that contain Prhl(RL)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL. <br>  
−
We saw that our new ''rhl(RL)'' promoter was actually activated by C4HSL through an induction assay (Fig. 3).<br>
+
We saw that our new Prhl(RL) promoter was actually activated by C4HSL through an induction assay. (Fig. 3.)<br>
  
 
[[Image:Improved_Prhl_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 3.</b> Fluorescence intensity detected by flow cytometer]]
 
[[Image:Improved_Prhl_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 3.</b> Fluorescence intensity detected by flow cytometer]]

Revision as of 02:22, 14 October 2014

Prhl(RL)-GFP

Prhl(RL) is a promoter that is activated by C4HSL.
We improved Prhl (BBa_R0071) by changing LuxR binding site of Plux (BBa_R0062) to RhlR binding site. (Fig.1 .)
The bottom structures of LuxR and RhlR are similar to each other, so RhlR can also bind to Lux Box. (Fig. 2.)

On the downstream of the promoter, GFP(BBa_J54103) is inserted as a reporter.


Fig. 1. Our newly designed promoter
Fig. 2. Difference between LuxR and RhlR

To characterize the function of the Prhl(RL) promoter (BBa_K1529300), we constructed this part, Prhl(RL)_GFP (BBa_K1529301), by inserting Prhl(RL) promoter upstream of a GFP coding sequence.
By using reporter cells that contain Prhl(RL)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL.
We saw that our new Prhl(RL) promoter was actually activated by C4HSL through an induction assay. (Fig. 3.)

Fig. 3. Fluorescence intensity detected by flow cytometer



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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 90
    Illegal BamHI site found at 78
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 777