Difference between revisions of "Part:BBa K1497019"

Revision as of 21:40, 11 October 2014

fdeR (Naringenin binding protein) and fde operon regulatory domain

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

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

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.

In vivo charakterisation 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 promotor (BBa_K1497017). They used the naringenin biosensor with GFP response K1497020 to characterise the naringenin biosynthesis operon in E. coli BL21(DE3).

The result are shown in figure 4. The GFP fluorescene is only the cells with the T7 naringenin operon visible and detectable. The team detemine 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 theE. coli BL21(DE3)-pSB1C3-fdeR-gfp strain containing and not containing the T7-Naringenin operon.

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

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

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 <td style="padding: 0cm 5.4pt; vertical-align: top; width: 306.7pt; height: 214.9pt;">
-<b>Naringenin</b> is the mayor flavonoe from grapefruits. In plants, it is synthesized from tyrosine and is one of the centrale metabolite in the flavonoe biosynthesis.It is able to reduce the oxdiative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are envoled in the degardation of coffein and increase the effect of coffein after the inhibition with naringenin.
+<b>Naringenin</b> is the mayor flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolite in the flavone biosynthesis. It is able to reduce the oxdiative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are involved in the degradation of caffeine and increase the effect of caffeine after the inhibition with naringenin.
 <br><br>
 <b>FdeR</b> is a homo dimeric protein from <i>Herbaspirillum seropedicae</i>. 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  <i>Herbaspirillum seropedicae</i> the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enable the growth with naringenin and the naringenin chalcone.