Revision as of 15:12, 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 control of an 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

Table. 1: ethylene detected quantities.

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.

B. subtilis

[http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Ethylene&action=edit Edit this page] | Main Page

@@ Line 18: / Line 18: @@
 		<img style="box-shadow:none;"src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" />
-		<h2>E.coli</h2>
+		<h2>E. coli</h2>
                  <img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts"/><br/>
@@ Line 89: / Line 89: @@
 		<div class="separator"></div>
+		<h2>B. subtilis</h2>
+		<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
 	</div>
 </div>
 <!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/3/3d/Tn-2013-headerbg-Sfondowm.jpg</html>|<html>https://static.igem.org/mediawiki/2013/1/10/Tn-2013-headerbgSfondowm_or.jpg</html>}}