Team:UNITN-Trento/Project/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 intermediate molecule (Goto M., Plant and Cell Physiology 2012, 26: 141-150).

We characterized this gene in two chassis: E. coli and B. subtilis, using different constructs that we designed.

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.

Ethylene production was detected using a Micro Gas Chromatograph (see the protocol page for the adopted methodology, Figure 1). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).

To quantify the amount of ethylene produced the peak integral was converted into ppm.

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

Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.

A toxicity test was performed inducing EFE expression with 5 mM arabinose (Figure 3). The growth curve was then compared to a non-induced sample.

As expected, induced samples showed a decreased growth rate.

To build our final system we placed EFE under the control of a photoinducible circuit. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala 2011 and Berkeley 2006). The construct BBa_K1065311 includes an inverter that allows ethylene production only in presence of light. For more details on the design and characterization of the circuit check the blue light page of our wiki.

We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065311, Figure 4). At an O.D. of 0.7, the culture was transferred to an hermetically closed vial and exposed to a blue light LED (470 nm). This airtight vial was also connected to the micro GC (see the protocol page for the adopted method).

In order to transform B. subtilis with EFE, we decided to exploit two type of vectors designed by the LMU-Munich 2012 iGEM team: pXyl and pSpac. These two vectors were not functionally active: pXyl had a point mutation resulting in a non-transformable vector, and pSpac had a point mutation in the promoter resulting in a non-inducible but constitutive vector. We received from the LMU-Munich team the corrected and functionally active version of both plasmids (functionality was characterized by them).

We successfully created and transformed the parts shown above (see B. subtilis page for details) but we did not detect any amount of ethylene with the Micro Gas Chromatograph.

Different fruits need different concentrations of ethylene when threated for triggering the ripening process. When we created our ripen machine device, we also though to do an estimation of the concentration of ethylene that there would be in the jar. To do this, we assumed that there was proportionality between air volume / culture volume ratio adopted. Since in the ethylene detection experiment we used a ratio equal to 4 and detected about 101 &plusmin; ppm, we simply applied a proportion to calculate the expected ethylene concetration in the different jars adopted in the experiments (500 ml, 1000 ml and 1500 ml). To threat fruit, we used 300 ml of culture in a 500 ml beute (so with 200 ml of air to add to the jar volume + 100 ml for the air in the connection).

In a second step, we performed a kinetic assay for ethylene diffusion in the jar (1000 ml and 1500 ml), exploting special caps with two connectors (one for the culture and one for the Micro Gas Chromatograph), taking a measure every 45 min for about 12 h.

2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:

• EFE was expressed under the control of an arabinose inducible promoter in E. coli;
• ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;
• EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production;
• EFE was expressed in B. subtilis under the control of two different inducible promoters. Ethylene was not detected but induced samples showed a particular smell. Further analysis demostrated that induced samples reacted with lead-acetate paper strips, indicating the presence of sulfur compounds. Probably B. subtilis is capable of converting rapidly ethylene in other compounds;
• successfully demonstrated and quantified the presence of ethylene in the jars;
• our system was successfully exploited to accelerate fruit ripening.