Team:UNITN-Trento/Project/Ethylene

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 (Goto M., Plant and Cell Physiology 2012, 26: 141-150).

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

EFE in 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. Ethylene detection

Ethylene production 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. 1: Ethylene production. Cells transformed with BBa_K1065001 were grown in a thermoshaker until an O.D. of 0.5, placed in hermetically closed vial with a rubber septum and induced with 5 mM Arabinose. Ethylene was measured after 4 hours of induction at 37 °C by connecting the vial to a Agilent Micro GC 3000.

To quantify the amount of ethylene produced the peak integral was converted into 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. 2. 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. 2: ethylene production (ppm) over time (min) of cells transformed with BBa_K1065001 and induced with 5 mM Arabinose at different O.D.600 and cultured in different conditions. The control (not-induced sample) did not show any amount of ethylene.

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

3. Toxicity test

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

Fig. 3: growth curves of cells transformed with BBa_K1065001 and of controls.

As expected, induced samples showed a decreased growth rate.

EFE under the control of a Blue light circuit in E. coli

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 (Uppsala2011 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 anc characterization of the circuit check the Blue light page of our wiki.

Photoinduced ethylene production - kinetic assay

We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065311). When the culture reached an OD of 0.7, it was placed in a hermetically closed vial and 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) upon photoinduction with a blue led light over time (min) of cells transformed with BBa_K1065311. EFE in B. subtilis

In order to tranform 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 recieved from the LMU-Munich team the corrected and functionally active version of both plasmids (functionality prooved by them).

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

Ethylene experiments - Summary

2-Oxoglutarate Oxygenase/Decarboxylase (EFE), is a very powerfull enzyme that has been successfully characterized. At the end of a set of experiments 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;
• our system was succcessfully exploited to accelerate fruit ripening.

Check how we exploited B. fruity to ripen fruit!