Difference between revisions of "Part:BBa K1497019"
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<partinfo>BBa_K1497019 short</partinfo>
 
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FdeR is a homo dimeric protein from ''Herbaspirillum seropedicae''. In present of naringenin, FdeR activates the specific promotor region upstream of the fdeR region and allow a strong gene expression.
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<b>Naringenin</b> is the mayor flavonoe from grapefruits and one of the centrale metabolite in the flavonoe biosynthesis. It is able to reduce the oxdiative stress and inhibit some P450 Enzymes.
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<b>FdeR</b> is a homo dimeric protein from ''Herbaspirillum seropedicae''. In present of naringenin (or naringenin chalchone), FdeR activates the specific promotor region upstream of the fdeR region and allow a strong gene expression. <br> In  ''Herbaspirillum seropedicae'' the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enable the growth with naringenin and the naringenin chalcone. 
  
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So in combination with GFP or an other fluorescense protein these part can be used as a in vivo naringenin sensor.
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  src="https://static.igem.org/mediawiki/2014/3/38/Naringeninsensensorschemagr%C3%BCn.png"></p>
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1</b></span></a><span lang="EN-US">
 
Flow chart of the FdeR activated gfp expression. The constitutive expression of fdeR forms the homodimeric FdeR protein. In present of naringenin, , naringenin molecules bind to the FdeR protein and operate a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now enabled to bind to the uncharacterized promotor domain.  Binding to the promotor domain induces expression of genes, which are cloned behind fdeR promtor region. </span></p>
 
Flow chart of the FdeR activated gfp expression. The constitutive expression of fdeR forms the homodimeric FdeR protein. In present of naringenin, , naringenin molecules bind to the FdeR protein and operate a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now enabled to bind to the uncharacterized promotor domain.  Binding to the promotor domain induces expression of genes, which are cloned behind fdeR promtor region. </span></p>
 
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  [[File:Homology_model_of_FdeR.png|300px|thumb|right|Homology model of FdeR. PDB: 2ESN was used as template. ]]
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Based on this reporter protein and 3 different fluorescence proteins, we designed 3 new biosensors for in vivo detection and determination of naringenin.
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You can use the reporters for measuring naringenin concentrations in your samples.  
 
You can use the reporters for measuring naringenin concentrations in your samples.  
 
Depending on which fluorophor you want to detect, you can use one of three biosensors:
 
Depending on which fluorophor you want to detect, you can use one of three biosensors:
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    <li>with GFP response use          <a href="/Part:BBa_K1497020">BBa_K1497020</a></li>
 
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    <li>with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
 
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    <li>with CFP response use         <a href="/Part:BBa_K1497022">BBa_K1497022</a></li>
 
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      <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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   <li>B: with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
 
   <li>B: with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
 
   <li>C: with no reporter              <a href="/Part:BBa_K1497019">BBa_K1497019</a></li>  
 
   <li>C: with no reporter              <a href="/Part:BBa_K1497019">BBa_K1497019</a></li>  
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   <li>D: with GFP response use          <a href="/Part:BBa_K1497020">BBa_K1497020</a></li>
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   <li>D: with GFP response use          <a href="/Part:BBa_K1497020">BBa_K1497020</a></li>  
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      <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick <html><a href="/Part:BBa_K1497019">K1497019</a></html> and clone your parts of interest (without RBS!) behind the device.
 
You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick <html><a href="/Part:BBa_K1497019">K1497019</a></html> and clone your parts of interest (without RBS!) behind the device.
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===Functional Parameters===
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The Biobrick <html><a href="/Part:BBa_K1497019">BBa_K1497019</a></html> produce in E. coli B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 mesured the fluorescense of GFP and mKate after the incubation with diffrent conentrations of naringenin. The results are shown in Figure 3. 
  
 
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===Functional Parameters===
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<img src="https://static.igem.org/mediawiki/2014/1/12/Naringenint7balken.png" align="right"
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iGEM TU Darmstadt 2014 used the naringenin biosensor with GFP response to characterise the naringenin biosynthesis in E. coli BL21(DE3)
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 21:41, 10 October 2014

fdeR (Naringenin binding protein) and fde operon regulatory domain



Naringenin is the mayor flavonoe from grapefruits and one of the centrale metabolite in the flavonoe biosynthesis. It is able to reduce the oxdiative stress and inhibit some P450 Enzymes.

FdeR is a homo dimeric protein from ''Herbaspirillum seropedicae''. In present of naringenin (or naringenin chalchone), FdeR activates the specific promotor region upstream of the fdeR region and allow a strong gene expression.
In ''Herbaspirillum seropedicae'' the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enable the growth with naringenin and the naringenin chalcone. So in combination with GFP or an other fluorescense protein these part can be used as a in vivo naringenin sensor.


Figure 1 Flow chart of the FdeR activated gfp expression. The constitutive expression of fdeR forms the homodimeric FdeR protein. In present of naringenin, , naringenin molecules bind to the FdeR protein and operate a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now enabled to bind to the uncharacterized promotor domain. Binding to the promotor domain induces expression of genes, which are cloned behind fdeR promtor region.



Usage and Biology

You can use the reporters for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors:




  • A: with CFP response use BBa_K1497022
  • B: with mKate response use BBa_K1497021
  • C: with no reporter BBa_K1497019
  • D: with GFP response use BBa_K1497020
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    Figure 2 E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample BBa_K1497019 without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

    You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.


    Functional Parameters

    The Biobrick BBa_K1497019 produce in E. coli B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 mesured the fluorescense of GFP and mKate after the incubation with diffrent conentrations of naringenin. The results are shown in Figure 3.


    Figure 3 Right: Characterization of BBa_K1497020. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 24 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well. Left: Characterization of BBa_K1497021. mKate (BBa_K1055000) fluorescence depends on the concentration of naringenin. We measured the mKate (BBa_K1055000) fluorescence after 24 h incubation with different concentrations of Naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.
    iGEM TU Darmstadt 2014 used the naringenin biosensor with GFP response to characterise the naringenin biosynthesis in E. coli BL21(DE3)

    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      COMPATIBLE WITH RFC[21]
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 95
      Illegal NgoMIV site found at 452
      Illegal NgoMIV site found at 543
      Illegal NgoMIV site found at 555
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 271