Results - Ethylene

EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyzes ethylene synthesis from 2-Oxoglutarate, a TCA cycle intemediate molecule. We characterized this gene in two chassis: E. coli and B. subtilis, using different contstructs that we designed.

E. coli

In E. coli, EFE-catalyzed ethylene production was characterized using BBa_K1065001, which is a composed part with EFE under the control of an AraC-pBAD promoter.

1. Toxicity test

A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a not-induced sample.

Fig. 1: growth curves of cells transformed with BBa_K1065001 and of controls.

As expected, induced samples showed a decreased growth rate.

2. Ethylene detection

Then, ethylene was detected using a Micro Gas Chromatograph (see the protocol page for the adopted methodology). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).

Fig. 2: chromatogram obtained using an Agilent Micro GC 3000A. A defined volume of culture was growth until his O.D. has reached 0.5. The cells were then induced with 5 mM Arabinose and putted into an ermetically closed vial with a piearciable septum. After 4 hours at 37 °C in thermoshaker, the samples were connected to the micro GC and a measure was taken.

The chromatogram clearly shows the presence of a peak corresponding to ethylene; the peak integral was converted to ppm.

Sample	Ethylene detected
Not induced	0 ± 15 ppm
Induced V = 1.5 ml	61 ± 15 ppm
Induced V = 3 ml	101 ± 15 ppm

Table. 1: ethylene detected quantities.

3. Kinetic assay for ethylene production

We performed a kinetic assay in order to analyze ethylene production over time (see the protocol page for the adopted method).

Fig. 3: ethylene production (ppm) over time (min) of cells induced at different O.D.600 and cultured at different environmental conditions.

Figure 3 shows how inducing the culture at O.D.600 equal to 0.8 a.u. caused a 2-fold increase in ethylene production.

E. coli - Blue light circuit

After having characterized Ethylene production using BBa_K1065001, we tried to put EFE under the control of a photoinducible circuit and we then tested his behaviour. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct includes an inverter that allows ethylene production only in presence of light.

Photoinduced ethylene production - kinetic assay

We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065XXX). The sample was then exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).

Fig. 4: ethylene production (ppm) over time (min) of cells induced at O.D.600 = 0.7 exposing the vial to a blue led (470 nm).

B. subtilis

Do you know how plants produce ethylene? Do you know that ethylene is used commercially to ripen some fruits before they enter the market?