Difference between revisions of "Part:BBa K1497022"

 
 
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<partinfo>BBa_K1497022 short</partinfo>
 
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We modified the naringenin biosensor from Siedler et al. by elimination of one EcoRI restriction site and by changing the native ribosomal binding site into the strong RBS Bba_B0034. Based on this reporter protein and 3 different fluorescence proteins, we designed 3 new biosensors for in vivo detection and determination of naringenin. The natural source of FdeR is Herbaspirillum seropedicae.
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You can use our reporter for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors:
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GFP fluorescence K1497020
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mKate (DsRed variant) fluorescence K1497021
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CFP fluorescence K1497022
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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/2014/9/9f/Naringeninsensensorschemagr%C3%BCnwnarin.png"></p>
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      <br>
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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 <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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===Usage and Biology===
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You can use the reporters for measuring naringenin concentrations in your samples.  
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Depending on which fluorophor you want to detect, you can use one of three biosensors:
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<br><br><br>
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<ul class="aufz10">
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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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src="https://static.igem.org/mediawiki/2014/8/89/Petridischnaringenin.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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<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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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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<partinfo>BBa_K1497022 parameters</partinfo>
 
<partinfo>BBa_K1497022 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 01:24, 18 October 2014

Naringenin sensor (FdeR) with CFP as reporter

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.



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