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Team:UNITN-Trento/Project/Ethylene
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Using <i>E. coli</i> as chassis, we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE. | Using <i>E. coli</i> as chassis, we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE. | ||
Revision as of 15:22, 26 August 2013
Results - Ethylene
EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses ethylene from 2-Oxoglutarate (a TCA cycle intemediate molecule).
We characterized that gene in two chassis: E. coli and B. subtilis using different contstructs that we designed.
Using E. coli as chassis, we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE.
1.Toxicity test
A toxicity test was done inducing cells to express EFE with 5mM Arabinose. A growth curve was then compared to a not-induced sample.
Fig. 1: a growth curve of cells transformed with BBa_K1065001.
As expected, induced saples showed a slowed but a not completely inhibited growth rate.
2.Ethylene detection
The next step was to detect ethylene using a Micro Gas Chromatograph (see the protocol page for method adopted).
Fig. 2: chromatogram obtained using an Agilent Micro GC 3000A.
The chromatogram clearly shows the presence of a peak corresponding to ethylene. The integral was then converted to ppm thanks to a calibration straight previously done using two different air mixtures (containing well-defined quantity of each molecule).
| 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
In order to analyze the production of ethylene during time, we performed a kinetic assay (see the protocol page for method adopted).
Fig. 3: plot rappresenting ethylene production during time.
As you can see from the plot, inducing culture at an O.D. of 0.8 caused an increase of more than 2 fold in ethylene.
Do you know how plants produce ethylene?Plants naturally produce ethylene starting from a common amino acid, methionine MET, wich is energized by ATP to produce S-adenosyl methionine SAM. The key enzyme in the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid ACC, the immediate precursor of ethylene. Quite recently researches purified the final enzyme in the pathway, ACC oxidase, wich converts ACC to ethylene.
All fruits, with a few exceptions (European pears, avocados and bananas), reach their best easting 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 postharvest handling system when shipped long-distance. Bananas for example are threated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Ripening of avocado, kiwifruit, mango and pear fruits before marketing is increasingly being used 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.
