Difference between revisions of "Part:BBa K1413001"

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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-well 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 of different concentrations Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
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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 of different concentrations Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
 
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Figure 1 describes the 96-wells plate organisation used to evaluate the biosensor.
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We used  three control in this experiment :<br/>
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Media only : To evaluate the natural fluorescence of the media with different concentrations of phenol.<br/>
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pSB1C3 : DH5alpha resistant to chloramphenicol used as a growth control.<br/>
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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.<br/>
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Purified GFP :  Used to associated fluorescence values to a defined concentration of GFP.<br/>
  
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<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/9/98/Bba_001_fluo_.jpg" width:"100px" width="500px;" class="thumbimage"/></div>
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    <div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/2/28/Plan_plaque_001_002.png" width:"100px" width="500px;" class="thumbimage"/></div>
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        <div align="center"><u>Figure 1 : 96-wells plate organisation scheme</u></div><br/>
  
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<br/> <div align="center"><u>Figure 1 Fluorescence intensity per cell of BBa_K1413001</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.
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<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/e/e2/EVRY_OD600_001.jpg"  width:"100px" width="500px;" class="thumbimage"/></div>
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      <div align="center"><u>Figure 2 : OD600 values measured over 11h in 96-wells plate(TECAN).</u></div><br/>
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<div align="center"><img alt="IMAGE" src="https://static.igem.org/mediawiki/parts/9/98/Bba_001_fluo_.jpg" width:"100px" width="500px;" class="thumbimage"/></div>
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<div align="center"><u>Figure 3 Fluorescence intensity per cell of BBa_K1413001</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.
 
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Revision as of 21:50, 1 November 2014

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

This part consist 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 sequence, a IHF binding site and a sigma factor 54 binding site.
-RBS 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.


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 of different concentrations Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).

Figure 1 describes the 96-wells plate organisation used to evaluate the biosensor. We used three control 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.

IMAGE
Figure 1 : 96-wells plate organisation scheme

IMAGE
Figure 2 : OD600 values measured over 11h in 96-wells plate(TECAN).

IMAGE
Figure 3 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.


IMAGE

Figure 1 Fluorescence intensity per cell of BBa_K1413001 and BBa_K1413002
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.


IMAGE

Figure 2 : Fluorescence induction ratio of BBa_K1413001 and BBa_K1413002
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)


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