Difference between revisions of "Part:BBa K1413001"

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<partinfo>BBa_K1413001 short</partinfo>
 
<partinfo>BBa_K1413001 short</partinfo>
 
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<br/> This part consists on a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family.  
+
<br/><br/>This part consists on a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family.  
 
Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the Po promoter, which drives transcription of  one single large operon for phenol degradation (dmpKLMNOPQBCDEFGHI).
 
Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the Po promoter, which drives transcription of  one single large operon for phenol degradation (dmpKLMNOPQBCDEFGHI).
 
With GFP attached to P0 promoter, it is then possible to evaluate the presence of phenol by fluorescence analysis, if DmpR is expressed.<br/>
 
With GFP attached to P0 promoter, it is then possible to evaluate the presence of phenol by fluorescence analysis, if DmpR is expressed.<br/>
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This part is basically composed of <br/><ul>
+
This part is basically composed of :<br/><ul>
 
<li>P0 promoter carrying two DmpR binding sites, a IHF binding site and a sigma factor 54 binding site.  
 
<li>P0 promoter carrying two DmpR binding sites, a IHF binding site and a sigma factor 54 binding site.  
 
<li>RBS <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>
 
<li>RBS <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>
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<li>Pr promoter, a constitutive promoter   
 
<li>Pr promoter, a constitutive promoter   
 
<li>DmpR the transcription factor that enable sensing of phenol.
 
<li>DmpR the transcription factor that enable sensing of phenol.
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</ul>
 
 
We characterized this biosensor using phenol as effector.  
 
We characterized this biosensor using phenol as effector.  
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+
</ul><b>Figure 1</b> describes the mechanism by which this biosensor respond to the presence of phenol.<br/>
 +
When DmpR binds to phenol its A domain uncovers the ATP binding site allowing it to bind to an ATP molecule.<br/>
 +
This binding triggers an hexamerisation of Dmpr dimers. This structure is then able to bind to DNA and activate transcriprtion of GFP. <br/><br/>
 
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     </div>
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     <center><u>Figure 1: Phenol construction and mechanism</u></center>
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     <center><u><b>Figure 1: Phenol construction and mechanism</b></u></center>
 
   </div>
 
   </div>
 
   </div>
 
   </div>
 
  </div>
 
  </div>
 
</div><br/>
 
</div><br/>
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When DmpR bind to phenol its A domain uncovers the ATP binding site allowing it to bind to an ATP molecule.<br/>
+
 
−
This binding triggers an hexamerisation of Dmpr dimers. This structure is then able to bind to DNA and activate transcriprtion of GFP. <br/>
+
 
</html>
 
</html>
  
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<br/>We prepared a protocol test to evaluate our Biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol at different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
 
<br/>We prepared a protocol test to evaluate our Biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol at different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
 
<br/><br/>
 
<br/><br/>
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Figure 2 describes the 96-wells plate organisation used to evaluate the biosensor.
+
<b>Figure 2 </b>  describes the 96-wells plate organisation used to evaluate the biosensor.
 
We used  four controls in this experiment :
 
We used  four controls in this experiment :
 
<ul>
 
<ul>
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<li><a href="https://parts.igem.org/Part:BBa_J23106">BBa_J23106</a> : DH5alpha carrying <a href="https://parts.igem.org/Part:BBa_J23106">BBa_J23106</a>, allowing constitutive production of GFP. This control was used to evaluate the eventual impact of phenol on GFP expression and/or fluorescence.
 
<li><a href="https://parts.igem.org/Part:BBa_J23106">BBa_J23106</a> : DH5alpha carrying <a href="https://parts.igem.org/Part:BBa_J23106">BBa_J23106</a>, allowing constitutive production of GFP. This control was used to evaluate the eventual impact of phenol on GFP expression and/or fluorescence.
 
<br/>
 
<br/>
−
<li>Purified GFP :  Used to associated fluorescence values to a defined concentration of GFP.</ul><br/>  
+
<li>Purified GFP :  Used to associated fluorescence values to a defined concentration of GFP.</ul>
 +
OD600 and Fluorescence intensity measured in plate are described in figure 3 and 4 respectively.<br/>  
 
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     </a>
 
     </a>
 
     </div>
 
     </div>
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     <center><u>Figure 2 : 96-wells plate organisation scheme</u></center>
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     <center><u><b>Figure 2 : 96-wells plate organisation scheme</b></u></center>
 
   </div>
 
   </div>
 
   </div>
 
   </div>
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     </a>
 
     </a>
 
     </div>
 
     </div>
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     <center><u>Figure 3 : OD600 values measured over 11h in 96-wells plate(TECAN)</u></center>
+
     <center><u><b>Figure 3 : OD600 values measured over 11h in 96-wells plate(TECAN)</b></u></center>
 
   </div>
 
   </div>
 
   </div>
 
   </div>
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     </a>
 
     </a>
 
     </div>
 
     </div>
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    <center><u>Figure 4 : Fluorescence intensity per cell of <a href="https://parts.igem.org/Part:BBa_K1413001">BBa_K1413001</a></u><br/> TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.</u></center>
+
  <center><u><b>Figure 4 : Fluorescence intensity per cell of BBa_K1413001.</b></u><br/> TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.</center>
 
   </div>
 
   </div>
 
   </div>
 
   </div>
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</div>
 
</div>
 
<br/><br/>
 
<br/><br/>
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These data show an increase in expression of sfGFP in response to increasing concentration of phenol present in wells.They also show that the biosensor is able to sense down to 1µM of phenol.
+
These data show an increase in expression of sfGFP in response to increasing concentration of phenol present in wells.They also show that the biosensor is able to sense down to 1µM of phenol<b>(Figure 4)</b>.
−
The induction ratio calculated from these data(Figure 4) show an 8-fold increase of fluorescence at 1µM of phenol and up to 45-fold increase at 1000µM.  
+
The induction ratio calculated from these data <b>(Figure 5)</b> show an 8-fold increase of fluorescence at 1µM of phenol and up to 45-fold increase at 1000µM.  
 
<br/><br/>
 
<br/><br/>
 
<div class="center">
 
<div class="center">
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     </a>
 
     </a>
 
     </div>
 
     </div>
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     <center><u>Figure 5 : Fluorescence induction ratio of <a href="https://parts.igem.org/Part:BBa_K1413001">BBa_K1413001</a></u><br/>TECAN  measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added)</u></center>
+
     <center><u><b>Figure 5 : Fluorescence induction ratio of BBa_K1413001.</b></u><br/>TECAN  measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added</center>
 
   </div>
 
   </div>
 
   </div>
 
   </div>
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</div>
 
</div>
 
<br/><br/>
 
<br/><br/>
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Comparing these data with those obtained by iGEM Peking 2013 indicates an improvement in the ability to produce a distinctive signal in response to phenol.<br/>(Figure 6)<br/>
+
Comparing these data with those obtained by iGEM Peking 2013 indicates an improvement in the ability to produce a distinctive signal in response to phenol.<br/><b>(Figure 6)</b><br/>
  
−
<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/e/e6/Peking_induction_ratio.PNG" width:"100px" width="300px;" class="thumbimage"/>  <img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/8/89/Evry_induction_log.jpg" width:"100px" width="320px;" class="thumbimage"/></div>
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<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/e/e6/Peking_induction_ratio.PNG" width:"100px" width="300px;" class="thumbimage"/>  <img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/archive/8/89/20141102201226!Evry_induction_log.jpg" width:"100px" width="320px;" class="thumbimage"/></div>
  
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<div align="center"><u>Figure 6 :Comparison of Phenol biosensors</u><br/> Left : Fluorescence induction ratio of Peking 2013 biosensor. Green curve corresponding phenol sensing.<br/> Right : Fluorescence induction ratio of BBak1413001 in response to phenol.
+
<div align="center"><u>Figure 6 :Comparison of Phenol biosensors</u><br/> Left : Fluorescence induction ratio of Peking 2013 biosensor. Green curve correspond to phenol sensing.<br/> Right : Fluorescence induction ratio of BBak1413001 in response to phenol.
 
</div><br/><br/>
 
</div><br/><br/>
  
−
<b>Improvement of <a href="https://parts.igem.org/Part:BBa_K1413001">BBa_K1413001</a> response to phenol.</b><br/>
+
<b>Improvement of BBa_K1413001 response to phenol.</b><br/>
−
We decided to strenghten the signal produced by our biosensor by mutating the ribosome binding site of sfGFP.
+
We decided to strengthen the signal produced by our biosensor by mutating the ribosome binding site of sfGFP.
 
This mutation is processed in a way that it reproduces the consensus sequence of Shine Dalgarno (AGGAGGUAA)allowing mRNA to bind more specifically to 16s rRNA. This in turn increases the translation rate of the mRNA.<br/><br/>
 
This mutation is processed in a way that it reproduces the consensus sequence of Shine Dalgarno (AGGAGGUAA)allowing mRNA to bind more specifically to 16s rRNA. This in turn increases the translation rate of the mRNA.<br/><br/>
 
See <a href="https://parts.igem.org/Part:BBa_K1413002">BBa_K1413002</a> part.<br/><br/>
 
See <a href="https://parts.igem.org/Part:BBa_K1413002">BBa_K1413002</a> part.<br/><br/>

Latest revision as of 22:11, 2 November 2014

P0 promoter-RBS B0032-sfGFP- Terminator B0015 - Pr promoter-DmpR

This part consists on a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family. Found in Pseudomonas sp. strain CF 600, DmpR regulates expression of the Po promoter, which drives transcription of one single large operon for phenol degradation (dmpKLMNOPQBCDEFGHI). With GFP attached to P0 promoter, it is then possible to evaluate the presence of phenol by fluorescence analysis, if DmpR is expressed.
This part is basically composed of :

  • P0 promoter carrying two DmpR binding sites, a IHF binding site and a sigma factor 54 binding site.
  • RBS BBa_B0032
  • The super folded GFP (sfGFP)
  • Pr promoter, a constitutive promoter
  • DmpR the transcription factor that enable sensing of phenol. We characterized this biosensor using phenol as effector.
Figure 1 describes the mechanism by which this biosensor respond to the presence of phenol.
When DmpR binds to phenol its A domain uncovers the ATP binding site allowing it to bind to an ATP molecule.
This binding triggers an hexamerisation of Dmpr dimers. This structure is then able to bind to DNA and activate transcriprtion of GFP.

IMAGE
Symbol
Figure 1: Phenol construction and mechanism

Usage and Biology


We prepared a protocol test to evaluate our Biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-wells plates. After 6 hours of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol at different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).

Figure 2 describes the 96-wells plate organisation used to evaluate the biosensor. We used four controls in this experiment :

  • Media only : to evaluate the natural fluorescence of the media with different concentrations of phenol.
  • pSB1C3 : DH5alpha resistant to chloramphenicol used as a growth control.
  • BBa_J23106 : DH5alpha carrying BBa_J23106, allowing constitutive production of GFP. This control was used to evaluate the eventual impact of phenol on GFP expression and/or fluorescence.
  • Purified GFP : Used to associated fluorescence values to a defined concentration of GFP.
OD600 and Fluorescence intensity measured in plate are described in figure 3 and 4 respectively.
IMAGE
Symbol
Figure 2 : 96-wells plate organisation scheme


IMAGE
Symbol
Figure 3 : OD600 values measured over 11h in 96-wells plate(TECAN)


IMAGE
Symbol
Figure 4 : Fluorescence intensity per cell of BBa_K1413001.
TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.


These data show an increase in expression of sfGFP in response to increasing concentration of phenol present in wells.They also show that the biosensor is able to sense down to 1µM of phenol(Figure 4). The induction ratio calculated from these data (Figure 5) show an 8-fold increase of fluorescence at 1µM of phenol and up to 45-fold increase at 1000µM.

IMAGE
Symbol
Figure 5 : Fluorescence induction ratio of BBa_K1413001.
TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added


Comparing these data with those obtained by iGEM Peking 2013 indicates an improvement in the ability to produce a distinctive signal in response to phenol.
(Figure 6)
IMAGE IMAGE
Figure 6 :Comparison of Phenol biosensors
Left : Fluorescence induction ratio of Peking 2013 biosensor. Green curve correspond to phenol sensing.
Right : Fluorescence induction ratio of BBak1413001 in response to phenol.


Improvement of BBa_K1413001 response to phenol.
We decided to strengthen the signal produced by our biosensor by mutating the ribosome binding site of sfGFP. This mutation is processed in a way that it reproduces the consensus sequence of Shine Dalgarno (AGGAGGUAA)allowing mRNA to bind more specifically to 16s rRNA. This in turn increases the translation rate of the mRNA.

See BBa_K1413002 part.

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 1241
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1719
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1404
    Illegal BsaI.rc site found at 1945
    Illegal SapI.rc site found at 211
    Illegal SapI.rc site found at 2602