Difference between revisions of "Part:BBa K1529321"
 
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<partinfo>BBa_K1529321 short</partinfo>
 
<partinfo>BBa_K1529321 short</partinfo>
  
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Prhl(a) is a promoter that is activated by C4HSL (Fig. 1).<br>
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This part is Prhl(RR) promoter regulating GFP.
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On the downstream of the promoter, GFP is inserted as a reporter.<br>
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Prhl(RR) is a promoter that is activated by C4HSL-RhlR complex.  <br>
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We improved Prhl (<partinfo>BBa_R0071</partinfo>)  by changing a LuxR binding site of Plux (<partinfo>BBa_R0062</partinfo>)  to a RhlR binding site. (Fig. 1.)<br>  
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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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[[Image:titech2014_parts_rhlpromoter_main_Fig1.jpg|thumb|center|250px|<b>Fig. 1.</b> Our newly designed  promoter]]
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We constructed this part to characterize the function of the Prhl(RR) promoter (<partinfo>BBa_K1529310</partinfo>), by inserting Prhl(RR) promoter upstream of a GFP coding sequence. <br>
  
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To characterize the function of the <i>Prhl(a)</i> promoter (<partinfo>BBa_K1529320</partinfo>), we constructed this part Prhl(a)_GFP (<partinfo>BBa_K1529321</partinfo>) by inserting ''rhl(a)'' promoter upstream of a GFP coding sequence. By using reporter cells that contain Prhl(a)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL (Fig. 2). <br>
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[[Image:titech2014_parts_rhl_promoter_main_Fig1.png|thumb|center|550px|<b>Fig. 1.</b> Our newly designed  promoter]]
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We saw that our new ''rhl(a)'' promoter was actually activated by C4HSL through an induction assay (Fig. 3).<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]]
  
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[[Image:titech2014_parts_GFPassay_main_Fig2.jpg|thumb|center|310px|<b>Fig. 2.</b> Fluorescence intensity detected by flow cytometer]]
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By using reporter cells that contain Prhl(RR)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL. <br>
 +
Through an induction assay, we found that our new Prhl(RL) promoter was actually activated by C4HSL-RhlR complex and had high induced/not-induced ratio(Fig. 3.). <br>
 +
This means Prhl(RL) promoter has the higher expression level while keeping the low leakage. <br>
 +
 
 +
We can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level. <br>
 +
 
 +
 
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[[Image:Improved_Prhl(RR)_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 3.</b> Fluorescence intensity detected by flow cytometer]]
  
 
<br>
 
<br>
  
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For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].
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For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay our work in Tokyo_Tech 2014 wiki].
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===Contribution: Waseda 2020===
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For the crosstalk experiments, we constructed two kinds of cell-free translation systems containing substantial amount of R proteins (LuxR,RhlR). In those systems, we put signaling molecules (3OHSL-C4 or 3OHSL-C6) and target genes (Plux/tet-GFP or Prhl-RR-GFP), respectively. We measured the fluorescence of GFP expressed from plux/tet-GFP and prhl(RR)-GFP on a RT-qPCR machine.
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Fig 1 shows the result of the crosstalk test in a cell-free system containing sufficient amount of LuxR protein.   In the results, non-cognate Prhl(RR) promter was highly activated by luxR-3OC6HSL complex as well as the cognate Plux/tet promoter.
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Fig 2 shows the result of the crosstalk test in a cell-free system containing sufficient amount of RhlR protein. As a result, we revealed that Prhl(RR)-GFP can work in vitro.
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[[Image:T--Waseda--crosstalk_luxR.png|thumb|center|600px|<b>Fig. 1. </b>Crosstalk assay in a cell-free system containing LuxR protein.
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]]<br>
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[[Image:T--Waseda--crosstalk_rhlR.png|thumb|center|600px|<b>Fig. 2. </b>Crosstalk assay in a cell-free system containing rhlR protein.
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]]<br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:33, 27 October 2020

Prhl(RR)_GFP

This part is Prhl(RR) promoter regulating GFP. Prhl(RR) is a promoter that is activated by C4HSL-RhlR complex.
We improved Prhl (BBa_R0071) by changing a LuxR binding site of Plux (BBa_R0062) to a 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.)

We constructed this part to characterize the function of the Prhl(RR) promoter (BBa_K1529310), by inserting Prhl(RR) promoter upstream of a GFP coding sequence.

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


By using reporter cells that contain Prhl(RR)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL.
Through an induction assay, we found that our new Prhl(RL) promoter was actually activated by C4HSL-RhlR complex and had high induced/not-induced ratio(Fig. 3.).
This means Prhl(RL) promoter has the higher expression level while keeping the low leakage.

We can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.


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].

Contribution: Waseda 2020

For the crosstalk experiments, we constructed two kinds of cell-free translation systems containing substantial amount of R proteins (LuxR,RhlR). In those systems, we put signaling molecules (3OHSL-C4 or 3OHSL-C6) and target genes (Plux/tet-GFP or Prhl-RR-GFP), respectively. We measured the fluorescence of GFP expressed from plux/tet-GFP and prhl(RR)-GFP on a RT-qPCR machine.

Fig 1 shows the result of the crosstalk test in a cell-free system containing sufficient amount of LuxR protein.   In the results, non-cognate Prhl(RR) promter was highly activated by luxR-3OC6HSL complex as well as the cognate Plux/tet promoter. Fig 2 shows the result of the crosstalk test in a cell-free system containing sufficient amount of RhlR protein. As a result, we revealed that Prhl(RR)-GFP can work in vitro.

Fig. 1. Crosstalk assay in a cell-free system containing LuxR protein.

Fig. 2. Crosstalk assay in a cell-free system containing rhlR protein.


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