Difference between revisions of "Part:BBa K1497021"
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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. | <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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| − | <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 | + | <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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| − | + | 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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Revision as of 00:52, 18 October 2014
Naringenin sensor (FdeR) with mKate as reporter
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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 constitutive expression of fdeR the FdeR proteins form homodimers. In the presence of naringenin, naringenin molecules bind to the FdeR homodimer 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 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:
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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 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 diffrent 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. |
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 271