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 16 h of incubation 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 the cell transformed with BBa_K1497017 exhibited GFP fluorescence.

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

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.

In addition, we proved the expression of GFP by running a SDS-PAGE (12,5% polyacrylamide gel).

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 8 & 9.

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.10) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 11).

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

Futhermore, We engineered an improved anthocyanidin synthase (eANS) by using rational design methods.

Production

To analyze the pelargonidin 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-beta-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9 media and fermented for 48h at 30°C in the presence of 0.1mM naringenin.

Extraction

After the production of pelargonidin producing operon with eANS (K1497015) in the presence of 0.5mM narigenin, we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet. We verified the pH-dependency of pelargonidin color (Fig. 13).

Construction of Grätzel cells

We used the extracted Pelargonidin (produced from E. coli BL21 (DE3) containing the pelargonidin producing operon, K1497015) to build a Grätzel cell. This was done according to Michael Grätzel & Brian O'Reagan. We were able to produce a voltage of 380 mV and a current 0.5 mA under daylight conditions.