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Team:UNITN-Trento/Project/Ethylene
From 2013.igem.org
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Revision as of 14:10, 16 September 2013
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 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.
Fig. 2: chromatogram obtained using an Agilent Micro GC 3000A.
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 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.
