"
Team:UNITN-Trento/Project/Ethylene
From 2013.igem.org
Results - Ethylene
EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses 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 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 curve of cells transformed with BBa_K1065001 and of the 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.
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 |
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 cultures 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 an increase of more than 2 fold in ethylene production.
Do you know how plants produce ethylene?Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed S-adenosyl-methionine (SAM) by a reaction catalyzed by SAM synthetase. The key enzyme of the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Quite recently, researchers purified the final enzyme of the pathway: ACC oxidase, which converts ACC to ethylene.
All fruits, with a few exceptions (European pears, avocados and bananas), reach their best eating quality when allowed to ripen on the tree or plant. However, some fruits are usually picked mature but unripe so that they can withstand the post-harvest handling system when shipped long-distance. Bananas, for example, are treated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Treatment of avocado, kiwi fruit, mango and pear fruits before marketing is increasingly being performed to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruit that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.
