Team:UNITN-Trento/Project/Ethylene

From 2013.igem.org

(Difference between revisions)
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<span class="tn-title">Results - Ethylene</span>
<span class="tn-title">Results - Ethylene</span>
<p>
<p>
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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 <span class="tn-ref">(Goto M., Plant and Cell Physiology 2012, 26: 141-150)</span>.
+
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 intermediate molecule <span class="tn-ref">(Goto M., Plant and Cell Physiology 2012, 26: 141-150)</span>.
</p>
</p>
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<p>
<p>
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We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different contstructs that we designed.
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We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.
</p>
</p>
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<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" />
<p>
<p>
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In <i>E. coli</i>, EFE-catalyzed ethylene production was characterized using <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, which is a composed part with EFE under the control of an AraC-pBAD promoter.
+
In <i>E. coli</i>, EFE-catalyzed ethylene production was characterized using <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, which is a composed part with EFE under the control of an araC-pBAD promoter.
</p>
</p>
<span class="tn-sub-subtitle">Ethylene detection</span>
<span class="tn-sub-subtitle">Ethylene detection</span>
<p>
<p>
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Ethylene production was detected using a Micro Gas Chromatograph (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#ethylene-detection-assay">protocol page</a> 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).
+
Ethylene production was detected using a Micro Gas Chromatograph (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#ethylene-detection-assay">protocol page</a> for the adopted methodology, <b>Figure 1</b>). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).
</p>
</p>
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" />
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<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> 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.</span>
+
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> 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 an Agilent Micro GC 3000.</span>
<p>
<p>
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<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" />
</div>
</div>
-
<span class="tn-caption"><b>Fig. 2:</b> ethylene production (ppm) over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> 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.</span>
+
<span class="tn-caption"><b>Fig. 2:</b> ethylene production (ppm) over time (min) of <i>E. coli</i> cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> 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.</span>
<p>
<p>
-
Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.
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<b>Figure 2</b> shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.
</p>
</p>
<span class="tn-sub-subtitle">Toxicity test</span>
<span class="tn-sub-subtitle">Toxicity test</span>
<p>
<p>
-
A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a non-induced sample.
+
A toxicity test was performed inducing EFE expression with 5 mM arabinose (<b>Figure 3</b>). The growth curve was then compared to a non-induced sample.
</p>
</p>
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<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" />
<p>
<p>
-
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 <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> 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.
+
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 (Uppsala 2011 and Berkeley 2006). The construct <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> includes an inverter that allows ethylene production only in presence of light. For more details on the design and characterization of the circuit check the blue light page of our wiki.
</p>
</p>
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span>
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span>
<p>
<p>
-
We performed a kinetic assay in order to analyze ethylene production over time using (<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>). 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).
+
We performed a kinetic assay in order to analyze ethylene production over time using (<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>, <b>Figure 4</b>). At an O.D. of 0.7, the culture was transferred to an hermetically closed vial and exposed to a blue light LED (470 nm). This airtight vial was also connected to the micro GC (see the protocol page for the adopted method).
</p>
</p>
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<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" />
</div>
</div>
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<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span>
+
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span>
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span>
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span>
<p>
<p>
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In order to tranform <i>B. subtilis</i> with EFE, we decided to exploit two type of vectors designed by the <a href="https://2012.igem.org/Team:LMU-Munich/Data/Vectors">LMU-Munich 2012 iGEM team</a>: 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 <b>corrected and functionally active version of both plasmids</b> (functionality prooved by them).
+
In order to transform <i>B. subtilis</i> with EFE, we decided to exploit two type of vectors designed by the <a href="https://2012.igem.org/Team:LMU-Munich/Data/Vectors">LMU-Munich 2012 iGEM team</a>: 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 received from the LMU-Munich team the <b>corrected and functionally active version of both plasmids</b> (functionality was characterized by them).
</p>
</p>
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" />
<p>
<p>
-
We successfully created and transformed the parts showed above (see <a><i>B. subtilis</i> page</a> for details) but we did not detect any amount of ethylene with the Micro Gas Chromatograph.
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We successfully created and transformed the parts shown above (see <a><i>B. subtilis</i> page</a> for details) but we did not detect any amount of ethylene with the Micro Gas Chromatograph.
</p>
</p>
<span class="tn-subtitle">Ethylene diffusion in jars</span>
<span class="tn-subtitle">Ethylene diffusion in jars</span>
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<p>
+
<p>
-
Different fruits need different concentrations of ethylene when threated for triggering the ripening process. When we created our ripen machine device, we also though to do an estimation of the concentration of ethylene that there would be in the jar. To do this, we assumed that there was proportionality between air volume / culture volume ratio adopted. Since in the ethylene detection experiment we used a ratio equal to 4 and detected about 101 &plusmin; ppm, we simply applied a proportion to calculate the expected ethylene concetration in the different jars adopted in the experiments (500 ml, 1000 ml and 1500 ml). To threat fruit, we used 300 ml of culture in a 500 ml beute (so with 200 ml of air to add to the jar volume + 100 ml for the air in the connection).
+
Different fruits need different concentrations of ethylene when threated for triggering the ripening process. When we created our ripen machine device, we also though to do an estimation of the concentration of ethylene that there would be in the jar. To do this, we assumed that there was proportionality between air volume / culture volume ratio adopted. Since in the ethylene detection experiment we used a ratio equal to 4 and detected about 101 &plusmin; ppm, we simply applied a proportion to calculate the expected ethylene concetration in the different jars adopted in the experiments (500 ml, 1000 ml and 1500 ml). To threat fruit, we used 300 ml of culture in a 500 ml beute (so with 200 ml of air to add to the jar volume + 100 ml for the air in the connection).  
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</p>
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</p>
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<table>
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<table>
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<tr>
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<tr>
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<th>
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<th>
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Jar volume (ml)
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Jar volume (ml)
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</th>
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</th>
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<th>
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<th>
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Air volume (ml)
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Air volume (ml)
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</th>
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</th>
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<th>
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<th>
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Culture volume (ml)
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Culture volume (ml)
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</th>
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</th>
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<th>
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<th>
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Air/culture volumes ratio
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Air/culture volumes ratio
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</th>
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</th>
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<th>
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<th>
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Concentration Expected
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Concentration Expected
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</th>
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</th>
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</tr>
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</tr>
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<tr>
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<tr>
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<td>
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<td>
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500
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500
-
</td>
+
</td>
-
<td>
+
<td>
-
800
+
800
-
</td>
+
</td>
-
<td>
+
<td>
-
300
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300
-
</td>
+
</td>
-
<td>
+
<td>
-
2.66
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2.66
-
</td>
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</td>
-
<td>
+
<td>
-
150.37
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150.37
-
</td>
+
</td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td>
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<td>
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1000
+
1000
-
</td>
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</td>
-
<td>
+
<td>
-
1300
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1300
-
</td>
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</td>
-
<td>
+
<td>
-
300
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300
-
</td>
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</td>
-
<td>
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<td>
-
4.33
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4.33
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</td>
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</td>
-
<td>
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<td>
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92.37
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92.37
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</td>
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</td>
-
</tr>
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</tr>
-
<tr>
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<tr>
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<td>
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<td>
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1500
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1500
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</td>
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</td>
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<td>
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<td>
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1800
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1800
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</td>
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</td>
-
<td>
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<td>
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300
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300
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</td>
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</td>
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<td>
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<td>
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6
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6
-
</td>
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</td>
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<td>
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<td>
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66.66
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66.66
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</td>
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</td>
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</tr>
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</tr>
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</table>
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</table>
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<span class="tn-caption"><b>Table 2:</b>ethylene expected in jars.</span>
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<span class="tn-caption"><b>Table 2:</b>ethylene expected in jars.</span>
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<br/>
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<br/>
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<p>
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<p>
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In a second step, we performed a kinetic assay for ethylene diffusion in the jar (1000 ml and 1500 ml), exploting special caps with two connectors (one for the culture and one for the micro Gas Chromatograph), taking a measure every 45 min for about 12 h.
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In a second step, we performed a kinetic assay for ethylene diffusion in the jar (1000 ml and 1500 ml), exploting special caps with two connectors (one for the culture and one for the Micro Gas Chromatograph), taking a measure every 45 min for about 12 h.
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</p>
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</p>
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<img src="https://static.igem.org/mediawiki/2013/1/1d/Tn-2013_eth_diff_apparatus.JPG" style="display:inline-block;width:40%;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="photo"/>
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<img src="https://static.igem.org/mediawiki/2013/1/1d/Tn-2013_eth_diff_apparatus.JPG" style="display:inline-block;width:40%;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="photo"/>
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<img src="https://static.igem.org/mediawiki/2013/a/af/Ethylene_diffusion_in_jars.png" style="display:inline-block;width: 58%;height: 307px;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="plot"/>
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<img src="https://static.igem.org/mediawiki/2013/a/af/Ethylene_diffusion_in_jars.png" style="display:inline-block;width: 58%;height: 307px;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="plot"/>
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<span class="tn-caption"><b>Fig 5:</b> In the right panel a kinetic assay for ethylene diffusion in jars. 300 ml of culture where induced at O.D. 0.5 and placed into a 500 ml beute connected to a jar. Moreover the jar was connected to the Agilent A3000 micro GC. Every 45 min a measure was taken for about 12 h. The lines show the expected values. In the left panel, a picture of the experimental set up. </span>
+
<span class="tn-caption"><b>Fig 5:</b> In the right panel a kinetic assay for ethylene diffusion in jars. 300 ml of culture where induced at O.D. 0.5 and placed into a 500 ml beute connected to a jar. Moreover the jar was connected to the Agilent A3000 micro GC. Every 45 min a measure was taken for about 12 h. The lines show the expected values. In the left panel, a picture of the experimental set up. </span>
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<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span>
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<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span>
<span class="tn-title">Ethylene experiments - Summary</span>
<span class="tn-title">Ethylene experiments - Summary</span>
<p>
<p>
-
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:
+
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:
</p>
</p>
<ul>
<ul>
<li>
<li>
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EFE was expressed under the control of an Arabinose inducible promoter in <i>E. coli</i>;
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EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;
</li>
</li>
<li>
<li>
-
ethylene was detected at the micro gas chromatograph and a quantitative kinetic curve was registered;
+
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;
</li>
</li>
<li>
<li>
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</li>
</li>
<li>
<li>
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successfully prooved and quantify ethylene in the jars where fruit were treated;
+
successfully demonstrated and quantified the presence of ethylene in the jars;
</li>
</li>
<li>
<li>

Revision as of 12:35, 28 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 intermediate molecule (Goto M., Plant and Cell Physiology 2012, 26: 141-150).

Ethylene pathway

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

EFE in E. coli E. coli EFE parts

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.

Ethylene detection

Ethylene production was detected using a Micro Gas Chromatograph (see the protocol page for the adopted methodology, Figure 1). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).

Ethylene chromatogram 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 an 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. 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).

kinetic_EFE_plot
Fig. 2: ethylene production (ppm) over time (min) of E. coli 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.

Toxicity test

A toxicity test was performed inducing EFE expression with 5 mM arabinose (Figure 3). The growth curve was then compared to a non-induced sample.

Toxicity test plot 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 e.coli_bluelight-EFE_parts

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 (Uppsala 2011 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 and 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, Figure 4). At an O.D. of 0.7, the culture was transferred to an hermetically closed vial and exposed to a blue light LED (470 nm). This airtight vial was also connected to the micro GC (see the protocol page for the adopted method).

EFE-blue_light_plot
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 transform 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 received from the LMU-Munich team the corrected and functionally active version of both plasmids (functionality was characterized by them).

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

Ethylene diffusion in jars

Different fruits need different concentrations of ethylene when threated for triggering the ripening process. When we created our ripen machine device, we also though to do an estimation of the concentration of ethylene that there would be in the jar. To do this, we assumed that there was proportionality between air volume / culture volume ratio adopted. Since in the ethylene detection experiment we used a ratio equal to 4 and detected about 101 &plusmin; ppm, we simply applied a proportion to calculate the expected ethylene concetration in the different jars adopted in the experiments (500 ml, 1000 ml and 1500 ml). To threat fruit, we used 300 ml of culture in a 500 ml beute (so with 200 ml of air to add to the jar volume + 100 ml for the air in the connection).

Jar volume (ml) Air volume (ml) Culture volume (ml) Air/culture volumes ratio Concentration Expected
500 800 300 2.66 150.37
1000 1300 300 4.33 92.37
1500 1800 300 6 66.66
Table 2:ethylene expected in jars.

In a second step, we performed a kinetic assay for ethylene diffusion in the jar (1000 ml and 1500 ml), exploting special caps with two connectors (one for the culture and one for the Micro Gas Chromatograph), taking a measure every 45 min for about 12 h.

Fig 5: In the right panel a kinetic assay for ethylene diffusion in jars. 300 ml of culture where induced at O.D. 0.5 and placed into a 500 ml beute connected to a jar. Moreover the jar was connected to the Agilent A3000 micro GC. Every 45 min a measure was taken for about 12 h. The lines show the expected values. In the left panel, a picture of the experimental set up. Interestingly, we underextimated the ethylene level in the jars! Ethylene experiments - Summary

2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. 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;
  • successfully demonstrated and quantified the presence of ethylene in the jars;
  • our system was successfully exploited to accelerate fruit ripening.

Check how we exploited B. fruity to ripen fruit!
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