Difference between revisions of "Part:BBa K1497022"

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:

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 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

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