We created the naringenin biosynthesis operon under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . We measured the naringenin production after a 16 h incubation time with the naringenin biosensor BBa_K1497020.

The cell pellets from E. coli BL21(DE3) - pSB1C3-fdeR-gfp with and without T7-naringenin operon (BBa_K1497017) are shown in figure 3. Only in the cell pellet with BBa_K1497017 exhibited GFP fluorescence.

We were also able to measure the GFP fluorescence quantitatively and to calculate with the help of a calibration curve for the naringenin sensor the production yield of both operons (Figure 4). For BBa_K1497017 we calculated 3 µM naringenin and for the operon with the constitutive promoter BBa_J23100 (BBa_K1497016) we calculated 1.9 µM naringenin.

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 Parameter

The Biobrick BBa_K1497019 produces in E. coli B and K strains the FdeR Protein. We measured the fluorescense of GFP and mKate after the incubation with different concentrations of naringenin. The results are shown in Figure 7 & 8.

Pelargonidin is an anthocyanin. Anthocyanins are water-soluble vacuolar pigments that appear yellow to dark-blue (pH-dependent), which are responsible for color of flowers and fruits and are health-promoting for humans.

We constructed a pelargonidin producing operon under the control of a T7 promoter (K1497015). The operon consists of 3 genes (flavonon-3beta-hydroxylase, dihydroflavonol 4-reductase, anthocyanindin synthase) each with strong RBS (Fig.1) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 2).

The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided from Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E. coli coding optimized and synthezised by Eurofins MWG opreon.

Futhermore, we designed two different ANS gene version by using XX to verify XXX

Production

To analyze the pelagonidin production operon (K1497015), we transformed it into E.coli Bl21 (DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan et al., (2007). After the induction with 1mM Isopropyl-?-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9 media and fermented for 48h at 37°C in present of 0.1mM naringenin.

Extraction

After the expression of pelagonidin producing operon with engineered ANS (K1497015) in present of 0.5mM narigenin we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet and supernatant and verified the pH-dependency of pelargonidin (Fig. 3).

Construction of Grätzel cells

We used the extracted Pelargonidin (produced from E.coli Bl21 (DE3) containing the pelagonidin producing operon, K1497015) to build a Grätzel cell. This was done according to Michael Grätzel & Brian O'Reagan. We were able to produce voltage of 380 mV and 0.5 mA under daylight conditions.