Difference between revisions of "Part:BBa K1497020"

Revision as of 01:22, 12 October 2022

Naringenin sensor (FdeR) with GFP 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.

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.

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 theE. coli BL21(DE3)-pSB1C3-fdeR-gfp strain containing and not containing the T7-Naringenin operon.

Improvement in 2022 (For Gold)

At this year's iGEM competition, team SHSBNU_China developed synthetic pathways for delphinidin, one particular kind of anthocyanin. Seven enzymes are needed to produce delphinidin. One of the intermediates is naringenin, a common plant extract substance. All intermediates, including the naringenin, are colorless and onerous to observe using a simple and on-the-spot method. Therefore, a working biosensor that can report fluorescence upon detecting the naringenin would be helpful.

In 2014, iGEM team TU_Darmstadt used to develop a naringenin sensor, documented here on this page.

The sensor will produce fluorescence upon receiving the naringenin signal, but unfortunately, after we synthesized this naringenin detector and constructed it on the PETDuet-1 plasmid, we failed to see GFP fluorescence signal even using 50 μM standard naringenin. PETDuet-GFP was used as a control to show at the right panel. We closely studied their sequence and referred to the original research paper. We realized their results were correct and great, but they made a typo mistake when uploading the sequence to the server. The GFP sequence should be on the opposite side of the activator protein in principle, but in the released sequence, the GFP and the FdeR regulator are on the same side. This will silence the GFP because the FdeR will only activate genes in opposite directions.

Fig. 1: The working principle of a naringenin sensor using FdeR protein as the regulator.

Fig. 2: The previously upload FdeR regulator does not produce a GFP signal upon adding naringenin 50uM.

Fig. 3: Our construction of the new expression system.

Fig. 4: Expression of GFP verified by SDS-Page with the new naringenin sensor and FdeR regulator.

Using the new plasmid， we tested whether it could detect naringenin. E.coli BL21 was transformed and we added naringenin into the LB liquid culture. However， no fluorescence was observed. We further lysed bacteria and conducted SDS-PAGE. Surprisingly, there was an expression band for GFP. We thought the naringenin detector was activated by naringenin, but the fluorescence seemed too weak. In the future, we will replace the GFP gene with other stronger GFP genes for more tests.

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