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 main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolites in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes is involved in the degradation of caffeine and increases the effect of caffeine after the inhibition with naringenin. 
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<br><br>
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<b>FdeR</b> is a homo dimeric protein from <i>Herbaspirillum seropedicae</i>. In the presence of naringenin (or naringenin chalcone), FdeR activates the specific promoter region upstream of the fdeR region and induces a strong gene expression. <br> In  <i>Herbaspirillum seropedicae</i> the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enables the growth with naringenin and the naringenin chalcone. 
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<br><br>
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When GFP or another reporter protein is cloned downstream of this part, it can be used as an <i>in vivo</i> 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 2</b></span></a><span lang="EN-US">
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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">
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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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Flow chart of the FdeR activated <i>gfp</i> expression. The constitutively expressed the FdeR monomers form homodimers. Naringenin molecules bind to the FdeR homodimer and induce a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now able to bind to the uncharacterized promoter region.  Binding to the promoter region induces expression of genes downstream of the fdeR promoter 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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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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  <li class="block-10vi">A: with CFP response use         <a href="/Part:BBa_K1497022">BBa_K1497022</a></li>
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  <li class="block-10vi">B: with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
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  <li class="block-10vi">C: with no reporter              <a href="/Part:BBa_K1497019">BBa_K1497019</a></li>
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  <li class="block-10vi">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">
 
       <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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  <i>E. coli</i> 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 fluorophor. 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>A: with CFP response use         <a href="/Part:BBa_K1497022">BBa_K1497022</a></li>
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  <li>B: with mKate response use <a href="/Part:BBa_K1497021">BBa_K1497021</a></li>
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  <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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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> produces in <i>E. coli</i> B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 measured the fluorescense of GFP and mKate after the incubation with different conentrations of naringenin. The results are shown in Figure 3. 
  
 
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 3</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 3</b></span></a><span lang="EN-US">
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<b>Right:</b> Characterization of  <a href="/Part:BBa_K1497020">BBa_K1497020</a>. 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. <b>Left:</b> Characterization of <a href="/Part:BBa_K1497021">BBa_K1497021</a>. mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence depends on the concentration of naringenin. We measured the mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence after 24 h incubation with different concentrations of Naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.</span></p>
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<b>Left:</b> Characterization of  <a href="/Part:BBa_K1497020">BBa_K1497020</a>. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well. <b>Right:</b> Characterization of <a href="/Part:BBa_K1497021">BBa_K1497021</a>. mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence depends on the concentration of naringenin. We measured the mKate (<a href="/Part:BBa_K1055000">BBa_K1055000</a>) fluorescence after 16 h incubation with different concentrations of Naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.</span></p>
 
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===Functional Parameters===
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====In vivo characterisation of the naringenin biosynthesis operon (<html><a href="/Part:BBa_K1497007">BBa_K1497007</a></html>)====
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iGEM TU Darmstadt 2014 reconstitute the naringenin biosynthesis in <i>E. coli</i> by construction of a operon polycistronic gene cluster (<a href="/Part:BBa_K1497007">BBa_K1497007</a>) under control of the strong T7 promoter (<a href="/Part:BBa_K1497017">BBa_K1497017</a>). They used the naringenin biosensor with GFP response <a href="/Part:BBa_K1497020">K1497020</a> to characterize the naringenin biosynthesis operon in <i>E. coli</i> BL21(DE3). <br><br> The result are shown in figure 4. The GFP fluorescene is only in the cells with the T7 naringenin operon visible and detectable. The team determined for this operon a naringenin production yield of 3 µmol naringenin per liter.   
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      <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 4</b></span></a><span lang="EN-US">
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<b>Left: </b>Cell pellets with and without T7-Naringenin operon from <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i>. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence. <b>Right: </b>Measurement of the GFP fluorescence in the<i> E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i> strain containing and not containing the T7-Naringenin operon.
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===Usage and Biology===
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===Usage and Biology===<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 characterize the naringenin biosynthesis operon (<a href="/Part:BBa_K1497017">BBa_K1497017</a>) in <i>E. coli</i> BL21(DE3). 
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<partinfo>BBa_K1497019 parameters</partinfo>
 
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====References====
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1. Siedler S, Stahlhut SG, Malla S, et al. (2014) Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. Metabolic engineering 21:2–8. doi: 10.1016/j.ymben.2013.10.011
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2. Fuhr UWE, Klittich K, Staib AH (1993) Inhibitory effect of grapefruit juice and its bitter principal, naringenin, on CYP1A2 dependent metabolism of caffeine in. Br J clin Pharmac 35:431–436.
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3. Marin a M, Souza EM, Pedrosa FO, et al. (2013) Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1. Microbiology (Reading, England) 159:167–75. doi: 10.1099/mic.0.061135-0

Latest revision as of 01:15, 18 October 2014

fdeR (Naringenin binding protein) and fde operon regulatory domain


Naringenin is the main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolites in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes is involved in the degradation of caffeine and increases the effect of caffeine after the inhibition with naringenin.

FdeR is a homo dimeric protein from Herbaspirillum seropedicae. In the presence of naringenin (or naringenin chalcone), FdeR activates the specific promoter region upstream of the fdeR region and induces a strong gene expression.
In Herbaspirillum seropedicae the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enables the growth with naringenin and the naringenin chalcone.

When GFP or another reporter protein is cloned downstream of this part, it can be used as an in vivo naringenin sensor.


Figure 1 Flow chart of the FdeR activated gfp expression. The constitutively expressed the FdeR monomers form homodimers. Naringenin molecules bind to the FdeR homodimer and induce a conformational change of the homodimeric FdeR structure. This conformational change activates FdeR, which is now able to bind to the uncharacterized promoter region. Binding to the promoter region induces expression of genes downstream of the fdeR promoter 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:




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 fluorophor. 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 produces in E. coli B and K strains the FdeR Protein. The iGEM Team TU Darmstadt 2014 measured the fluorescense of GFP and mKate after the incubation with different conentrations of naringenin. The results are shown in Figure 3.


Figure 3 Left: Characterization of BBa_K1497020. GFP fluorescence depends on the concentration of naringenin. We measured the GFP fluorescence after 16 h incubation with different concentrations of naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of GFP as well. Right: Characterization of BBa_K1497021. mKate (BBa_K1055000) fluorescence depends on the concentration of naringenin. We measured the mKate (BBa_K1055000) fluorescence after 16 h incubation with different concentrations of Naringenin. By setting higher concentrations of naringenin, we gained higher fluorescence of mKate as well.



In vivo characterisation of the naringenin biosynthesis operon (BBa_K1497007)


iGEM TU Darmstadt 2014 reconstitute the naringenin biosynthesis in E. coli by construction of a operon polycistronic gene cluster (BBa_K1497007) under control of the strong T7 promoter (BBa_K1497017). They used the naringenin biosensor with GFP response K1497020 to characterize the naringenin biosynthesis operon in E. coli BL21(DE3).

The result are shown in figure 4. The GFP fluorescene is only in the cells with the T7 naringenin operon visible and detectable. The team determined for this operon a naringenin production yield of 3 µmol naringenin per liter.



Figure 4 Left: Cell pellets with and without T7-Naringenin operon from E. coli BL21(DE3)-pSB1C3-fdeR-gfp. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence. Right: Measurement of the GFP fluorescence in the E. coli BL21(DE3)-pSB1C3-fdeR-gfp strain containing and not containing the T7-Naringenin operon.


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



References

1. Siedler S, Stahlhut SG, Malla S, et al. (2014) Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. Metabolic engineering 21:2–8. doi: 10.1016/j.ymben.2013.10.011

2. Fuhr UWE, Klittich K, Staib AH (1993) Inhibitory effect of grapefruit juice and its bitter principal, naringenin, on CYP1A2 dependent metabolism of caffeine in. Br J clin Pharmac 35:431–436.

3. Marin a M, Souza EM, Pedrosa FO, et al. (2013) Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1. Microbiology (Reading, England) 159:167–75. doi: 10.1099/mic.0.061135-0