http://2013.igem.org/wiki/index.php?title=Special:Contributions/Xli&feed=atom&limit=50&target=Xli&year=&month=2013.igem.org - User contributions [en]2024-03-28T09:48:54ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UNITN-Trento/Project/EthyleneTeam:UNITN-Trento/Project/Ethylene2013-10-28T22:42:38Z<p>Xli: </p>
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited but then we found that the negative control (dark) also showed some ethylene production. DNA sequencing confirmed that there were some problems with this construct. We have now fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Importantly, the blue reporter correctly appeared only in the induced sample, so we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<a id="newGC"></a><span class="tn-sub-subtitle">New GC measurements on the circuit: we can control ethylene production in a non-chemical way!</span><br />
<br />
Finally between the European jamboree and the world championship we were able to take more measurements on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram"/><br />
<span class="tn-caption"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure (92 &plusmn;15 ppm), while it is not produced in the dark.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that the behavior of the circuit was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper.<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /><br />
<span class="tn-caption"><b>Fig. 7:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (37 &plusmn;15 ppm) instead in the presence of blue light there was no ethylene produced.</span><br />
<br />
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all. However the on/off switch was better defined with the circuit without an inverter.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 8:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underestimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into two different versions of a photoinducible circuit (with and without an inverter), GC measurements were taken and the results were consistent with the engineered function;<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
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We envision the construction of a genetic circuit that either produces ethylene or methyl-salicylate (MeSA) in response to either the presence or absence of light. In the dark state the sensor protein YF-1 first auto-phosphorylates and then phosphorylates the response regulator FixJ. Phosphorylated FixJ is then able to bind to the pFixK2 promoter thereby activating transcription of cI (the inhibitor protein) that ultimately blocks EFE production. At the same time, phosphorylated FixJ activates the transcription of Bsmt1, which initiates the synthesis of MeSA, thus slowing down fruit ripening. <br/><br/><br />
<br />
In the presence of blue-light, the sensor protein YF-1 does not auto-phosphorylate and does not phosphorylate FixJ. The RNA polymerase does not recognize the pFixK2 promoter. The result is that ethylene is produced and MeSA synthesis is blocked. In other words, fruit ripening is induced.<br />
</div><br />
<br />
<span class="tn-subtitle">These are our best characterized parts on the system</span><br />
<span class="tn-sub-subtitle"><b>Best natural part</b></span><br />
<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, 2-Oxoglutarate Oxygenase/Decarboxylase Ethylene Forming Enzyme (EFE). This part was well characterized under the control of different inducible promoters in both <i>E. coli</i> (NEB10beta cells) and in <i>B. subtilis</i> (str.168).<br />
<span class="tn-sub-subtitle"><b>Best engineered device</b><br></span><br />
<a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>, blue light regulated amilCP producing device. This device includes an inverter cassette (composed by cI protein and pLambda promoter) that ultimately allows production of the chromoprotein amilCP only when the culture is exposed to light.<br />
<span class="tn-sub-subtitle"><b>Best improved part</b><br></span><br />
<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, blue light regulated amilGFP producing device (a yellow fluorescent protein). The part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a>, extracted from the registry, was not working because it was missing the RBS between pFixK2 promoter and amilGFP coding sequence. We improved this part by mutagenesis and demonstrated that it is able to produce amilGFP in the dark. .<br/><br/><br />
<br />
<span class="subtitle">We also characterized the following parts:</span><br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>: methyl-salycilate producing device.<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>: an improved version of our methyl-salycilate producing device. This device include the SAM synthetase gene to boost wintergreen production.<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>: EFE under the control of the blue light circuit with inverter. This device produces ethylene upon photoinduction.<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_K1065309</a>: EFE under the control of the blue light circuit without inverter. This device successfully produced ethylene in the dark.<br />
</li><br />
</ul><br />
<br />
<span class="tn-effect-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripening">Check how we exploited our parts to ripen fruit!</a></span><br />
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited but then we found that the negative control (dark) also showed some ethylene production. DNA sequencing confirmed that there were some problems with this construct. We have now fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Importantly, the blue reporter correctly appeared only in the induced sample, so we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<a id="newGC"></a><span class="tn-sub-subtitle">New GC measurements on the circuit: we can finally control ethylene production in a non-chemical way!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measurements on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram"/><br />
<span class="tn-caption"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure, while it is not produced in the dark.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that the behavior of the circuit was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /><br />
<span class="tn-caption"><b>Fig. 7:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead in the presence of blue light there was no ethylene produced.</span><br />
<br />
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 8:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underestimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into two different versions of a photoinducible circuit (with and without an inverter), GC measurements were taken and the results were consistent with the engineered function;<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited but then we found that the negative control (dark) also showed some ethylene production. DNA sequencing confirmed that there were some problems with this construct. We have now fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Importantly, the blue reporter correctly appeared only in the induced sample, so we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<a id="newGC"></a><span class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measurements on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram"/><br />
<span class="tn-caption"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is produced. In the dark ethylene is not produced.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /><br />
<span class="tn-caption"><b>Fig. 7:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead in the present of blue light there was no ethylene produced.</span><br />
<br />
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 8:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underestimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into two different versions of a photoinducible circuit (with and without an inverter), GC measurements were taken and the results were consistent with the engineered function;<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
</div><br />
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited but then we found that the negative control (dark) also showed some ethylene production. DNA sequencing confirmed that there were some problems with this construct. We have now fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Importantly, the blue reporter correctly appeared only in the induced sample, so we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<a id="newGC"></a><span class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measure<ments on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram" /><br />
<span class="tn-caption"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is produced. In the dark ethylene is not produced.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /><br />
<span class="tn-caption"><b>Fig. 7:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead in the present of blue light there was no ethylene produced.</span><br />
<br />
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 8:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underestimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into two different versions of a photoinducible circuit (with and without an inverter), GC measurements were taken and the results were consistent with the engineered function;<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction"><br />
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<a id="tn-sp-tour" href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue%20light#tour"><br />
<img src="https://static.igem.org/mediawiki/2013/8/81/Tn-2013-tour-T_AAA_DSC_0076.png" /><br />
<span>Continue the tour!</span><br />
</a><br />
<br />
<!--end content--><br />
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<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Judging" class="news">We got gold and are on our way to MIT!! <img class="photo" src="https://static.igem.org/mediawiki/2013/b/b3/Tn-2013-Gold_award.png" /></a><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene#newGC" class="news-2">Check out our brand new results!!!</a><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-TrentoTeam:UNITN-Trento2013-10-28T12:53:27Z<p>Xli: </p>
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<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Judging" class="news">We got gold and we are on our way to MIT!!!<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b3/Tn-2013-Gold_award.png" /></a><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene#newGC" class="news-2">Check out our brand new results!!!</a><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/World_Championship_Jamboree/Practice_SessionsWorld Championship Jamboree/Practice Sessions2013-10-27T21:57:02Z<p>Xli: </p>
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<p class="contentheader">Practice Sessions - November 01</p><br />
<p>Use this sign-up sheet to sign up for a practice session slot on Friday night (<b>November 1st</b>) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. Be sure to bring necessary computer equipment with you, such as chargers and adapters, as these will not be provided.</p><br />
<br />
<p>There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room (please see the <a href="https://2013.igem.org/World_Championship_Jamboree/Map">campus map</a> for building locations). This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time.</p><br />
<br />
<p>Also, pre-registration will be available on Friday November 1st, starting at 3pm at <b>Building 12, Room 156</b>. Conference services will be on-site to pass out team registration boxes (see the <a href="https://2013.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a>).</p> <br />
<br />
<p><strong>Note</strong>: Use the wiki edit button to add your team to the schedule (the markup is located at the bottom of the page). Additional rooms may be added in the coming weeks.<br />
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<th style="width:100px;">Time</th><br />
<th>RM 32-123 </th><br />
<th>RM 32-141 </th><br />
<th>RM 32-155 </th><br />
<th>RM 34-101 </th><br />
<th>RM 56-114 </th><br />
<th>RM 56-154 </th><br />
<th>RM 66-144 </th><br />
<th>RM 66-168 </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>5:00 - 5:30PM</th><br />
<td>BIT-China</td><br />
<td><i>unavailable</i></td><br />
<td>SYSU-Software</td><br />
<td>ZJU-China</td><br />
<td>Valencia Biocampus</td><br />
<td>A6</td><br />
<td>A7</td><br />
<td>A8</td><br />
</tr><br />
<tr class="even"><br />
<th>5:30 - 6:00PM</th><br />
<td>Freiburg</td><br />
<td><i>unavailable</i></td><br />
<td>TU-Eindhoven</td><br />
<td>Tokyo_Tech</td><br />
<td>Bielefeld-Germany</td><br />
<td>B6</td><br />
<td>B7</td><br />
<td>B8</td><br />
</tr><br />
<tr class="even"><br />
<th>6:00 - 6:30PM</th><br />
<td>Dundee</td><br />
<td>UNITN-Trento</td><br />
<td>Göttingen</td><br />
<td>C4</td><br />
<td>C5</td><br />
<td>C6</td><br />
<td>C7</td><br />
<td>C8</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30 - 7:00PM</th><br />
<td>SJTU-BioX-Shanghai</td><br />
<td>D2</td><br />
<td>HUST-China</td><br />
<td>NJU China</td><br />
<td>D5</td><br />
<td>D6</td><br />
<td>D7</td><br />
<td>D8</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00 - 7:30PM</th><br />
<td>uOttawa</td><br />
<td>SYSU-China</td><br />
<td>XMU Software</td><br />
<td>Peking</td><br />
<td>E5</td><br />
<td>E6</td><br />
<td>E7</td><br />
<td>E8</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30 - 8:00PM</th><br />
<td>XMU China</td><br />
<td>Braunschweig</td><br />
<td>SUSTC-Shenzhen-B</td><br />
<td>NCTU_Formosa</td><br />
<td>F5</td><br />
<td>F6</td><br />
<td>F7</td><br />
<td>F8</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00 - 8:30PM</th><br />
<td>Calgary</td><br />
<td>G2</td><br />
<td>USTC-Software</td><br />
<td>NYMU-Taipei</td><br />
<td>UC_Davis</td><br />
<td>G6</td><br />
<td>G7</td><br />
<td>G8</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30 - 9:00PM</th><br />
<td>TU-Munich</td><br />
<td>ETH Zurich</td><br />
<td>Shenzhen_BGIC_0101</td><br />
<td>Virginia</td><br />
<td>H5</td><br />
<td>Tsinghua</td><br />
<td>H7</td><br />
<td>H8</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00 - 9:30PM</th><br />
<td>Tianjin</td> <br />
<td>Imperial College</td><br />
<td>Shenzhen_BGIC_ATCG</td><br />
<td>Paris_Bettencourt</td><br />
<td>Penn</td><br />
<td>Yale</td><br />
<td>Calgary_Entrepreneurial</td><br />
<td>UCSF</td><br />
</tr><br />
</tbody><br />
</table><br />
<br /><br />
<img src="https://static.igem.org/mediawiki/2013/f/f6/2013WCJ_practice_sessions_map.png" /><br />
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</html></div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/EthyleneTeam:UNITN-Trento/Project/Ethylene2013-10-26T16:54:50Z<p>Xli: </p>
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited. However, the negative control (dark) showed also some ethylene production. The DNA sequencing also indicated some problems in the construct. We have recently fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Since the blue reporter correctly appeared only in the induced sample, we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<span style="text-align:justify;"class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram" /><br />
<span class="tn-caption center"><b>Fig. 5:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is produced. In the dark ethylene is not produced.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /><br />
<span style="text-align:justify;" class="tn-caption center"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then splitted and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead with the blue light on there was no ethylene.</span><br />
<br />
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 7:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production (unfortunately also in the dark control);<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
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<br />
box-shadow: 2px 2px 4px #323232;<br />
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text-align: justify;<br />
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<br />
line-height: 1.2em;<br />
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}<br />
<br />
/*GRID*/<br />
<br />
.grid {<br />
width: 960px;<br />
margin: 0 auto;<br />
padding-bottom: 2em;<br />
}<br />
<br />
.column {<br />
display: inline-block;<br />
margin: 0;<br />
padding: 0;<br />
width: 315px;<br />
vertical-align: top;<br />
}<br />
<br />
.cell {<br />
display: inline-block;<br />
<br />
padding: 0;<br />
<br />
background-color: white;<br />
border: 3px solid white;<br />
box-shadow: 2px 2px 4px black;<br />
<br />
vertical-align: top;<br />
}<br />
<br />
.column.first .cell.first {<br />
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margin: 10px 7px 7px 10px;<br />
}<br />
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.column.first .cell.second {<br />
margin: 7px 7px 10px 10px;<br />
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background-image: url(https://static.igem.org/mediawiki/2013/9/97/Tn-2013-home-Fruit-ripening.png);<br />
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}<br />
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margin: 7px 10px 10px 0px;<br />
<br />
background-image: url(https://static.igem.org/mediawiki/2013/8/8c/Tn-2013-home-Know-your-fruit.jpg);<br />
}<br />
<br />
.column.first .cell.first,<br />
.column.first .cell.second,<br />
.column.third .cell.first,<br />
.column.third .cell.second {<br />
height: 222px;<br />
width: 293px;<br />
white-space: nowrap;<br />
}<br />
<br />
.column.first .cell.first:hover,<br />
.column.first .cell.second:hover,<br />
.column.third .cell.first:hover,<br />
.column.third .cell.second:hover {<br />
box-shadow: 2px 2px 4px #323232 inset;<br />
}<br />
<br />
.column.first .cell.first a,<br />
.column.first .cell.second a,<br />
.column.third .cell.first a,<br />
.column.third .cell.second a {<br />
display: block;<br />
<br />
height: 100%;<br />
width: 100%;<br />
}<br />
<br />
.column.second .cell.first {<br />
position: relative;<br />
<br />
margin: 10px 0px;<br />
height: 464px;<br />
width: 300px;<br />
<br />
background-color: rgba(245, 90, 0,.75);<br />
background-image: url( https://static.igem.org/mediawiki/2013/b/bf/Tn-2013_Onda-fruitripening.png);<br />
}<br />
<br />
.column.second .cell.first .tn-title {<br />
display: block;<br />
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color: white;<br />
font-family: "Boogaloo", cursive;<br />
font-size: 2.5em;<br />
text-align: center;<br />
text-shadow: 2px 2px 4px #323232;<br />
}<br />
<br />
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display: block;<br />
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margin: 1em auto;<br />
margin-left: 1em;<br />
padding: 10px;<br />
padding-left: 25px;<br />
<br />
<br />
width: 200px;<br />
<br />
background-color: rgba(255,255,255,.8);<br />
border-radius: 1em;<br />
<br />
font-size: 0.85em;<br />
line-height: 1em;<br />
list-style: disc;<br />
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margin-bottom: 0.3em;<br />
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<br />
.column.second .cell.first img {<br />
position: absolute;<br />
<br />
margin: 0.8em;<br />
width: 70px;<br />
}<br />
<br />
.column.second .cell.first img.plasmid {<br />
top: 95px;<br />
right: -10px;<br />
<br />
width: 65px;<br />
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<br />
.column.second .cell.first img.human {<br />
top: 190px;<br />
right: -10px;<br />
<br />
width: 65px;<br />
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<br />
.column.second .cell.first img.product {<br />
top: 270px;<br />
right: -10px;<br />
<br />
width: 65px;<br />
}<br />
<br />
.column.second .cell.first img.fruit {<br />
top: 380px;<br />
right: -10px;<br />
<br />
width: 65px;<br />
}<br />
<br />
/*POST-GRID*/<br />
<br />
.post-grid {<br />
position: relative;<br />
width: 960px;<br />
margin: 0 auto;<br />
}<br />
<br />
.post-grid .sheet {<br />
width: 922px;<br />
min-height: 100px;<br />
<br />
margin-top: -2em;<br />
margin-left: 10px;<br />
margin-bottom: 2em;<br />
<br />
background-color: rgba(39,48,77,.9);<br />
background-image: url( https://static.igem.org/mediawiki/2013/f/f6/Tn-2013-boxbg-Onda-Blueberry.png);<br />
border: 3px solid white;<br />
<br />
box-shadow: 2px 2px 4px #323232;<br />
<br />
text-align: justify;<br />
z-index: 1;<br />
}<br />
<br />
<br />
.post-grid p {<br />
margin: 0.4em auto;<br />
<br />
line-height: 1.2em;<br />
}<br />
<br />
.post-grid .txt {<br />
display: box;<br />
position: absolute;<br />
top: -0.5em;<br />
left: 0.7em;<br />
<br />
color: white;<br />
font-size: 3em;<br />
font-family: "Boogaloo", cursive;<br />
text-shadow: 0 0 6px black;<br />
<br />
z-index: 5;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-TrentoTeam:UNITN-Trento2013-10-26T14:11:15Z<p>Xli: </p>
<hr />
<div><!--NEW PAGE--><br />
<br />
{{:Team:UNITN-Trento/Templates/Default|<html><!--start content--><br />
<br />
<!-- CSS --><br />
<link rel="stylesheet" href="https://2013.igem.org/Team:UNITN-Trento/CSS/Home?action=raw&ctype=text/css" /><br />
<br />
<!-- JS --><br />
<script type="text/javascript" src="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/JS/Home&action=raw&ctype=text/javascript"></script><br />
<br />
<!-- PAGE --><br />
<div style ="width: 941px;margin: auto;margin-bottom: -70px;margin-top: -63px;"><span class="news" style="display:inline;">We obtained the gold medal and a ticket to Boston!!!</span><img style="height:200px;display:inline;" src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png"/></div><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-TrentoTeam:UNITN-Trento2013-10-26T14:04:49Z<p>Xli: </p>
<hr />
<div><!--NEW PAGE--><br />
<br />
{{:Team:UNITN-Trento/Templates/Default|<html><!--start content--><br />
<br />
<!-- CSS --><br />
<link rel="stylesheet" href="https://2013.igem.org/Team:UNITN-Trento/CSS/Home?action=raw&ctype=text/css" /><br />
<br />
<!-- JS --><br />
<script type="text/javascript" src="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/JS/Home&action=raw&ctype=text/javascript"></script><br />
<br />
<!-- PAGE --><br />
<div style ="width: 941px;margin: auto;margin-bottom: -70px;margin-top: -63px;"><span class="news" style="display:inline;">We obtained gold medal and a ticket to Boston!!!</span><img style="height:200px;display:inline;" src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png"/></div><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-TrentoTeam:UNITN-Trento2013-10-26T14:03:33Z<p>Xli: </p>
<hr />
<div><!--NEW PAGE--><br />
<br />
{{:Team:UNITN-Trento/Templates/Default|<html><!--start content--><br />
<br />
<!-- CSS --><br />
<link rel="stylesheet" href="https://2013.igem.org/Team:UNITN-Trento/CSS/Home?action=raw&ctype=text/css" /><br />
<br />
<!-- JS --><br />
<script type="text/javascript" src="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/JS/Home&action=raw&ctype=text/javascript"></script><br />
<br />
<!-- PAGE --><br />
<div style ="width: 941px;margin: auto;margin-bottom: -70px;margin-top: -63px;; margin:auto;"><span class="news" style="display:inline;">We obtained gold medal and a ticket to Boston!!!</span><img style="height:200px;display:inline;" src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png"/></div><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-TrentoTeam:UNITN-Trento2013-10-26T14:00:03Z<p>Xli: </p>
<hr />
<div><!--NEW PAGE--><br />
<br />
{{:Team:UNITN-Trento/Templates/Default|<html><!--start content--><br />
<br />
<!-- CSS --><br />
<link rel="stylesheet" href="https://2013.igem.org/Team:UNITN-Trento/CSS/Home?action=raw&ctype=text/css" /><br />
<br />
<!-- JS --><br />
<script type="text/javascript" src="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/JS/Home&action=raw&ctype=text/javascript"></script><br />
<br />
<!-- PAGE --><br />
<div style ="width:80px; margin:auto;"><span class="news" style="display:inline;">We obtained gold medal and a ticket to Boston!!!</span><img style="height:200px;display:inline;" src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png"/></div><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
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<span class="news">We obtained gold medal and a ticket to Boston!!!</span><img src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png"/><br />
<br />
<div class="pre-grid"><br />
<span class="silhouette"><br />
<img class="photo-1" src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013-silhouette.png"><br />
<img style="display: none;" class="photo-2" src="https://static.igem.org/mediawiki/2013/2/21/Tn-2013-silhouette-2.png" /><br />
</span><br />
<div class="sheet"><br />
<div class="container"><br />
<p>Hi everybody, UniTN-Trento team is proud to introduce you <i>B. fruity</i>!</p><br />
<br />
<p><i>B. fruity</i> envisions a new environmental friendly way to control fruit ripening by exploiting an engineered, light-regulated strain of <i>B. subtilis</i>. The system works by synthesizing ethylene or methyl salicylate (MeSA) upon photoinduction.</p><br />
<br />
<p><b>Ethylene</b> is a plant hormone widely used to ripen fruit. However its synthesis, handling and storage is expensive and dangerous. In contrast, <i>B. fruity</i> produces ethylene from inexpensive material by a metabolic intermediate: 2-oxoglutarate. Our system converts this metabolite to ethylene through the activity of an ethylene forming enzyme (EFE) from <i>Pseudomonas syringae</i>.</p><br />
<br />
<p><i>B. fruity</i> does not just accelerate ripening, but can also slow the process down, when desired, through the incorporation of a <b>methyl salicylate</b> (MeSA) synthesis pathway. MeSA was previously shown to inhibit the ripening of kiwifruit and tomatoes. The explored MeSA pathway builds upon the 2006 MIT iGEM project “<i>Eau de coli</i>”.</p><br />
<br />
<p>As a proof of concept, we engineered <i>E. coli</i> with the above system plus the YF1/FixJ blue light receptor device.</p><br />
<br />
<p>We are hopeful that <i>B. fruity</i> will simplify the process of bringing fresh fruit from the field to the consumer.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="grid"><br />
<div class="column first"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction#tour"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"></a><br />
</div><br />
</div><br />
<div class="column second"><br />
<div class="cell first"><br />
<span class="tn-title">Achievements</span><br />
<img class="plasmid" src="https://static.igem.org/mediawiki/2013/b/b6/Tn-2013-home-ach-Plasmids.png" /><br />
<ul class="plasmid"><br />
<li>Built and submitted 16 New BioBricks;</li><br />
<li>Successfully characterized 9 BioBricks;</li><br />
<li>Improved 4 parts;</li><br />
</ul><br />
<img class="human" src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-home-ach-Human-Practice_150.png" /><br />
<ul class="human"><br />
<li>Hosted a high school student in the lab for the summer;</li><br />
<li>Asked the opinion of local fruit consumers on our project;</li><br />
</ul><br />
<img class="product" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013-home-ach-Products.png" /><br />
<ul class="product"><br />
<li>Designed a fruit Vending Machine and a Home Edition <i>B. fruity</i> market product;</li><br />
</ul><br />
<img class="fruit" src="https://static.igem.org/mediawiki/2013/3/30/Tn-2013-home-ach-Fruit.png" /><br />
<ul class="fruit"><br />
<li>Ripened 5 bananas, 5 tomatoes, 3 kiwis, 2 plums, 2 apples;</li><br />
<li>Eaten one banana bread, one plums crumble and one apple strudel. </li><br />
</ul><br />
</div><br />
</div><br />
<div class="column third"><br />
<div class="cell first"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"></a><br />
</div><br />
<div class="cell second"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Extra/Fruit%20Info"></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/5/53/Tn-2013-headerbg-Sfondobb.png</html>|<html>https://static.igem.org/mediawiki/2013/d/d2/Tn-2013-headerbg-Sfondobb_or.png</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/EthyleneTeam:UNITN-Trento/Project/Ethylene2013-10-26T13:49:23Z<p>Xli: </p>
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited. However, the negative control (dark) showed also some ethylene production. The DNA sequencing also indicated some problems in the construct. We have recently fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Since the blue reporter correctly appeared only in the induced sample, we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<span style="text-align:justify;"class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="" alt="311_chromatogram" /><br />
<span class="tn-caption center"><b>Fig. 5:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is produced. In the dark ethylene is not produced.</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/><br />
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="" alt="309_chromatogram" /><br />
<span style="text-align:justify;" class="tn-caption center"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then splitted and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead with the blue light on there was no ethylene.</span><br />
<br />
However note that not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 7:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production (unfortunately also in the dark control);<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited. However, the negative control (dark) showed also some ethylene production. The DNA sequencing also indicated some problems in the construct. We have recently fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Since the blue reporter correctly appeared only in the induced sample, we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<span style="text-align:justify;"class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="" alt="311_chromatogram" /><br />
<span class="tn-caption center"><b>Fig. 5:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is way more than in the dark. We couldn’t see the complete shutdown of the system though since a little amount was produced in the dark too</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even in this case we observed some unfunctional colonies and some others producing ethylene in the control. <br/><br />
We also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="" alt="309_chromatogram" /><br />
<span style="text-align:justify;" class="tn-caption center"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then splitted and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead with the blue light on there was no ethylene.</span><br />
<br />
However note that not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 7:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production (unfortunately also in the dark control);<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
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<span class="tn-title">Results - Ethylene</span><br />
<p><br />
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>.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" /><br />
<br />
<p><br />
We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different constructs that we designed.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts" /><br />
<p><br />
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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Ethylene detection</span><br />
<p><br />
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).<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" /><br />
<span class="tn-caption"><b>Fig. 1:</b> Ethylene production. <i>E. coli</i> NEB10&beta; 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 vials 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><br />
<br />
<p><br />
To quantify the amount of ethylene produced the peak integral was converted into ppm.<br />
</p><br />
<br />
<table class="no-bottom" id="ethylene_detected"><br />
<tr><br />
<th class="center"><br />
Sample<br />
</th><br />
<th class="center"><br />
Ethylene detected<br />
</th><br />
</tr><br />
<tr><br />
<td><br />
Not induced<br />
</td><br />
<td class="right"><br />
0 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 1.5 ml<br />
</td><br />
<td class="right"><br />
61 ± 15 ppm<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Induced V = 3 ml<br />
</td><br />
<td class="right"><br />
101 ± 15 ppm<br />
</td><br />
</tr><br />
</table><br />
<br />
<span class="tn-caption center"><b>Table. 1:</b> ethylene detected quantities.</span><br />
<br />
<span class="tn-sub-subtitle">Kinetic assay for ethylene production</span><br />
<p><br />
We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2:</b> Ethylene production (ppm) over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, 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><br />
<br />
<p><br />
<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.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Toxicity test</span><br />
<p><br />
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.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" /><br />
<span class="tn-caption center"><b>Fig. 3:</b> growth curves of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.</span><br />
<br />
<p><br />
As expected, induced samples showed a decreased growth rate.<br />
</p><br />
<br />
<span class="tn-subtitle">EFE under the control of a Blue light circuit in <i>E. coli</i></span><br />
<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts" /><br />
<p><br />
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 <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue_light"> <b> blue light page </b> </a> of our wiki.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Photoinduced ethylene production - kinetic assay</span><br />
<p><br />
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).<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /><br />
<img class="photo no-bottom" style="height: 300px;" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.</span><br />
<br />
<br />
When we first obtained these results we were really excited. However, the negative control (dark) showed also some ethylene production. The DNA sequencing also indicated some problems in the construct. We have recently fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> is able to produce amilCP when photoinduced. Since the blue reporter correctly appeared only in the induced sample, we think that ethylene could be properly produced. <br><br/><br />
<br />
<img style="width:50%;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"/><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5: </b> amilCP production upon photoinduction. <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue LED. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white.</span> <br />
<br />
<span class="tn-sub-subtitle">New GC measures on the circuit!!!</span><br />
<br />
Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results.<br />
<br />
<img class="no-bottom" src="" alt="311_chromatogram" /><br />
<span class="tn-caption center"><b>Fig. 5:</b> <i>E. coli</i> NEB10&beta; tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is way more than in the dark. We couldn’t see the complete shutdown of the system though since a little amount was produced in the dark too</span><br />
<br />
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even in this case we observed some unfunctional colonies and some others producing ethylene in the control. <br/><br />
We also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results.<br />
<img class="no-bottom" src="" alt="309_chromatogram" /><br />
<span class="tn-caption center"><b>Fig. 6:</b> <i>E. coli</i> NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then splitted and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead with the blue light on there was no ethylene.</span><br />
<br />
However note that not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all.<br />
Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.<br />
<br />
<br />
<span class="tn-subtitle">EFE in <i>B. subtilis</i></span><br />
<p><br />
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).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg" /><br />
<p><br />
EFE was inserted in two <i>B. subtilis</i> plasmids under the control of two different inducible promoters. We tried to express EFE and measure ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme.<br />
<br />
Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds.<br />
</p><br />
<span class="tn-subtitle">Ethylene diffusion in jars</span><br />
<p><br />
Our ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in <b>Table 2</b>.<br />
</p><br />
<table><br />
<tr><br />
<th><br />
<center> Jar volume (ml) </center><br />
</th><br />
<th><br />
<center>Air volume in the jar + connections (ml) /<center><br />
</th><br />
<th><br />
<center> Culture volume (ml)</center><br />
</th><br />
<th><br />
<center> Air/culture volumes ratio </center><br />
</th><br />
<th><br />
<center> Concentration Expected </center><br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
<center> 500 </center> <br />
</td><br />
<td><br />
<center> 800 </center> <br />
</td><br />
<td><br />
<center> 300 </center> <br />
</td><br />
<td><br />
<center> 2.66 </center> <br />
</td><br />
<td><br />
<center> 150.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1000 </center> <br />
</td> <br />
<td><br />
<center>1300 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center> 4.33 </center> <br />
</td><br />
<td><br />
<center> 92.37 </center> <br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<center>1500 </center> <br />
</td> <br />
<td><br />
<center>1800 </center> <br />
</td><br />
<td><br />
<center>300 </center> <br />
</td><br />
<td><br />
<center>6 </center> <br />
</td><br />
<td><br />
<center>66.66 </center><br />
</td><br />
</tr><br />
</table> <br />
<span class="tn-caption center"><b>Table 2:</b> prediction of ethylene diffusion in jars.</span><br />
<br/><br />
<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"/><br />
<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"/><br />
<span class="tn-caption"><b>Fig 7:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span><br />
<br />
<span class="tn-quote center">Interestingly, we underextimated the ethylene level in the jars!</span><br />
<br />
<br />
<span class="tn-title">Ethylene experiments - Summary</span><br />
<p><br />
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:<br />
</p><br />
<ul><br />
<li><br />
EFE was expressed under the control of an arabinose inducible promoter in <i>E. coli</i>;<br />
</li><br />
<li><br />
ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;<br />
</li><br />
<li><br />
EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production (unfortunately also in the dark control);<br />
</li><br />
<li><br />
EFE was inserted into <i>B. subtilis</i> expression vectors, unfortunately ethylene was not detected upon expression;<br />
</li><br />
<li><br />
successfully demonstrated and quantified the presence of ethylene in the jars;<br />
</li> <br />
<li><br />
our system was successfully exploited to accelerate fruit ripening.<br />
</li><br />
</ul><br />
<br/><br />
<br />
<span class="tn-effect">We succeeded in producing ethylene with our system!</span><br />
<span class="tn-effect">Follow our results to discover how we used it to ripen fruit.</span><br />
<img style="width:60%;" src="https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg" /><br />
</div><br />
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<div></div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripeningTeam:UNITN-Trento/Project/Fruit ripening2013-10-15T12:56:00Z<p>Xli: </p>
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<br />
<span class="tn-title">Fruit ripening</span><br />
<p><br />
We have tested several type of fruit to verify that our engineered bacteria are able to produce <b>ethylene</b> (<i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>) and <b>methyl salicylate</b> (cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>) for enhancing or slowing down the ripening of fruit, respectively.<br />
</p><br />
<p><br />
The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator) where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in a airtight jar or a fruit placed in a jar connected to a flask with non-induced <i>E. coli</i> NEB10&beta; cells.<br />
</p><br />
<p><br />
Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: the same bunch of bananas).<br />
</p><br />
<p><br />
The results obtained varied according to the fruit tested. We tested: bananas, plums, cherries and heirloom tomatoes, blackberries, kiwifruit and others. Results were evaluated <b>qualitatively</b> by observing <b>skin color changes</b> and <b>pulp texture or firmness</b>.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/bd/Tn-2013_fruithood.jpg" /><br />
<p><br />
In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them.<br />
</p><br />
<p><br />
We were also able to perform a <b>iodine coloration test</b> on starch-containing fruit.<br />
</p><br />
<p><br />
We also performed <b>quantitative</b> evaluations of ethylene effects by <b>fruit compression strength test</b> and by a <b>fruit color measurement test</b>.<br />
</p><br />
<br />
<span class="tn-subtitle">Ethylene</span><br />
<p class="tn-msg">Click on the images to view them enlarged.</p><br />
<div id="jms-slideshow-1" class="jms-slideshow"><br />
<div id="ex1" class="step" data-scale="1" data-x="0" data-y="0" data-z="0"><br />
<p align="center"><br />
Cherry tomatoes are successfully ripened<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Cherry tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 8 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 1 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/7/7d/Tn-2013-fruit_pomodorini1.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div> <br />
<br />
<div id="ex2" class="step" data-scale="1" data-x="0" data-y="0" data-z="5"><br />
<p align="center"><br />
Ripened plums become red<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Plums<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 4 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 4 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 1 L jar<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/5/52/Tn-2013-fruit_susine1.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div id="ex3" class="step" data-scale="1" data-x="0" data-y="0" data-z="-10"><br />
<p align="center"><br />
Heirloom tomatoes ready to be eaten<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Heirloom tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 9 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 9 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 1 L jar<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/1/1b/Tn-2013-fruit_pomodorigrandi2.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div id="ex4" class="step" data-scale="1" data-x="0" data-y="0" data-z="-15"><br />
<p align="center"><br />
Cherry tomatoes are successfully ripened<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Cherry tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 7 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 1 day<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/2/24/Tn-2013-fruit_pomodorini2.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<div id="ex5" class="step" data-scale="1" data-x="0" data-y="0" data-z="-20"><br />
<p align="center"><br />
Cavendish bananas are successfully ripened<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Cavendish banana<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 6 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 6 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Essicator (10.3L)<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/3/39/Tn-2013-fruit-banana1.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<div id="ex6" class="step" data-scale="1" data-x="0" data-y="0" data-z="-25"><br />
<p align="center"><br />
"Fried Green Tomatoes"<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Date tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 7 days<br />
</p><br />
<p><br />
<span class="title">Exposition to Ethylene</span> 2 day w/, 3 days w/o, 2 days w/<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
<p><br />
Note: Lycopene synthesis probably inhibited by the high temperature (&gt;30 degrees) in the lab as suggested in the literature<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-fruit_datteri1.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<p><br />
In summary: <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that this experiment did not work because the temperature in the laboratory was over 30 degrees, causing a decreased activity of lycopene synthesis enzymes <span class="tn-ref">(Mary Lu Arpaia et Al., Fruit Ripening &amp; Ethylene Management 2010, 9-10)</span>.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Iodine Coloration Test</span><br />
<p><br />
Starch accumulates in apples during growing season and is hydrolyzed to sugar in the last stages of maturation and development <span class="tn-ref">(Krotkov and Helson 1946)</span>. Hydrolysis occurs first in the core area and progresses outwards <span class="tn-ref">(Phillips and Poapst 1952)</span>. Starch in cut sections of the fruit reacts with a solution of iodine-potassium iodide to produce a blue-black color. The intensity and pattern of the color developed is an indication of the stage of maturation.<br><br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013-fruit-Apple_starch_test.png" /><br />
<span class="tn-caption"><b>Figure 1:</b> Starch test on apples. An apple was exposed to a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> for three days and then left in a jar for other five days. It is well clear that treated apple is in a more advance ripening stage since iodine did not color the simple sugar produced from the hydrolisis of starch. Moreover we can assess a change in the pigment color (from green to red) of the treated apple.</span><br />
<br />
<span class="tn-sub-subtitle">Fruit Compression Strength Test</span><br />
<p><br />
A compression test was used to determine the maturation stage of kiwifruit and bananas. All these measurements were done in the laboratory of eng. Alessandro Pegoretti in the Industrial Engineering Department. In this test the fruit is subjected to a crushing load to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analyzed, as an indication of maturation stage.<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/e/ec/Tn-2013-fruit-Kiwi%27s_compressive_strength.png" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/a/a5/Tn2013_photkiwi.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Figure 2:</b> Results of the compression test on kiwifruit in the left panel. After exposure to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K651001</a> for 8 days the fruit were cut into small cubes of the same size. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with <a href="http://parts.igem.org/Part:BBa_K1065001">Bba_K1065001</a> in red), as demonstrated by the lower strength needed to compress the fruit. In the right panel the Instrom machinery used with a kiwi sample.</span><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/b/b0/Tn2013_tomphoto.JPG" /><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/9/9d/Tn-2013-fruit-Comparison_of_ultimate_strength_in_kiwi.png" /><br />
</div><br />
<span class="tn-caption"><b>Figure 3:</b> In the left panel Thomas is waiting for our results. In the right panel, averages of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and negative controls were left for 8 days in an airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based on the best 3 measurements obtained. A significant difference (25 fold less) between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripened the kiwifruit exposed to ethylene after 8 days.</span><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Banana%27s_compressive_strength.png" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_brunophoto.JPG" /><br />
</div><br />
<span class="tn-caption"><b>Figure 4:</b> In the left panel bananas compression test results. After exposure to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K651001</a> for 5 days the bananas were cut into small cubes of the same size. For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">Bba_K1065001</a> in red). In the right panel Bruno observes with curiosity the functioning of the machine.</span><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/6/65/Tn-2013_banana.JPG" /><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Comparison_of_ultimate_strength_in_banana.png" /><br />
</div><br />
<span class="tn-caption"><b>Figure 5:</b> In the left panel, a banana's sample between the plate of machinery. In the right panel average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and negative controls were left for 5 days in a airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. Treated bananas are 1.5 fold less firm than untreated bananas.</span><br />
<br />
<p><br />
The test helped us to quantify the stage of maturation of kiwifruit and bananas, as another confirmation that our ethylene producing device works as predicted. However the test worked better on kiwis rather than bananas probably because the bananas used started already from a more advanced maturation stage.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Fruit color measurement test</span><br />
<p><br />
We used this test to quantify the difference between the color of ripe and unripe fruit's pulp. All measurments were done in the laboratory of eng. Sara Spilimbergo in the Industrial Engineering Department.<br />
</p><br />
<p><br />
We estimated the color of kiwifruit using a high resolution miniature spectrometer, connected to a optical fiber.<br />
</p><br />
<p><br />
The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness). The L* parameter indicate a range between black (0) and white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow). A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/f/fc/Tn-2013-fruit-Lightness_of_kiwifruit.png" /><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/a/ad/Tn-2013_kiwi1.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> In the left panel lightness measurament of kiwifruit treated for 8 days with ethylene (<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> induced, green) and negative controls (No cells, blue and <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> not induced, red). Treated kiwifuit has a lower L* value as a proof of advanced stage of ripening. In the right panel the instrument used to acquired the data. We note the high resolution miniature spectrometer with an optic fiber reflection probe connected.</span><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kiwi2.JPG" /><br />
<img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013-fruit-Color_measurement_of_kiwifruit.png" /><br />
</div><br />
<span class="tn-caption"><b>Figure 7:</b> In the left panel a detail of the fiber optic reflection probe and a slice-sample of kiwifruit. In the right panel the color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> induced, blue) showed a consistent shift toward a darker green shade respect to controls (no cells, green and <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> not induced, red).</span><br />
<br />
<span class="tn-effect">We have engineered a device able to ripen different kind of fruit!</span><br />
<br />
<span class="tn-subtitle">Methyl salicylate</span><br />
<br />
<p class="tn-msg">Click on the images to view them enlarged.</p><br />
<div id="jms-slideshow-2" class="jms-slideshow"><br />
<div id="ex12" class="step" data-scale="1" data-x="0" data-y="0" data-z="0"><br />
<p align="center"><br />
Date tomatoes stay green<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Date tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 7 days<br />
</p><br />
<p><br />
<span class="title">Exposition to MeSA <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>)</span> 2 day w/, 3 days w/o, 2 days w/<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/3/3d/Tn-2013-fruit-datteri1_mesa.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<div id="ex22" class="step" data-scale="1" data-x="0" data-y="0" data-z="-5"><br />
<p align="center"><br />
Cherry tomatoes unripen<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Cherry tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 7 days<br />
</p><br />
<p><br />
<span class="title">Exposition to MeSA (<a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>)</span> 7 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
<p><br />
Note: One of the control was probably at a different maturation state ad day1.<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/8/87/Tn-2013-fruit-pomodorini2_mesa.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<div id="ex32" class="step" data-scale="1" data-x="0" data-y="0" data-z="-10"><br />
<p align="center"><br />
Pure MeSA has a toxic effect on cerry tomatoes<br />
</p><br />
<table><br />
<tr><br />
<td><br />
<p><br />
<span class="title">Type of fruit</span> Cherry tomatoes<br />
</p><br />
<p><br />
<span class="title">Duration of experiment</span> 8 days<br />
</p><br />
<p><br />
<span class="title">Immersion in MeSA pure</span> 8 days<br />
</p><br />
<p><br />
<span class="title">Container</span> Fruit in 0.5 L jar<br />
</p><br />
<p><br />
Note: The immersion in MeSA pure killed the fruit.<br />
</p><br />
</td><br />
<td><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013-fruit-pomodorini1_mesa.png"></a><br />
</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<p><br />
<i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> (our methyl salicylate producing devices) did not show any difference when compared to the negative control. We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.<br />
</p><br />
<p><br />
Exposure to pure MeSA coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana. MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM) <span class="tn-ref">(Ding, C. and Wang, Y. 164, Plant Science 2003, pp. 589-596)</span>. We think that under the used conditions its concentration was above the threshold required to inhibit ripening.<br><br />
</p><br />
<br />
<br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Blue%20light"><br />
<img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/d/d1/Tn-2013-arr-Ripening_prev.png" /><br />
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<img src="https://static.igem.org/mediawiki/2013/6/6e/Tn-2013-arr-AAA_TOP.png" /><br />
</a><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Bacillus"><br />
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</div><br />
<br />
<a id="tn-sp-tour" href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine#tour"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Tn-2013-tour-T_AAA-DSC_0050.png" /><br />
<span>Continue the tour!</span><br />
</a><br />
<br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Introduction</span><br />
<span class="tn-quote"><br />
Have you ever thrown away some bananas because they were too ripe?<br><br />
Are you one of many that hates waiting ages to eat the immature kiwis that you find at the supermarket?<br><br />
Do you know how bananas and other fruits are picked unripe from the tree and arrive to the supermarket ready to be sold and eaten?<br><br />
Have you ever thought about how much fruit is wasted in restaurants, markets, and industry?<br />
</span><br />
<p><br />
We have decided to solve both these problems by designing and engineering a bacterial system able to control fruit's ripening in response to different stimuli: <i>B. fruity</i>.<br />
</p><br />
<p><br />
Furthermore, we have planned two different commercial products in order to eliminate fruit waste and to make its consumption more accessible, even in unusual places like schools and offices: the "<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"><i>B. fruity </i>Vending Machine</a>" and the "<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Home%20Edition"><i>B. fruity </i>Home Edition</a>".<br />
</p><br />
<br />
<span class="tn-subtitle">How does <i>B. fruity </i> work?</span><br />
<p><br />
We designed and started to build a genetic circuit that allows our bacteria to synthesize ethylene in order to boost fruit ripening. Ethylene is an hormone naturally produced by fruit and it affects growth, development, ripening, and senescence <span class="tn-ref">(C. J. Brady, Plant Physiology 1987, 38: 155-178)</span>. However, we did not engineer <i>B. fruity</i> to use the complicated ethylene synthesis pathway present in plants, because of the undesirable production of hydrogen cyanide <span class="tn-ref">(Shang Fa Yang et Al., Plant Physiol. 2001, 126(2): 742–749.). </span> Instead, we decided to follow a different metabolic pathway, present in <i>Pseudomonas syringae</i>, which involves only one enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, an Ethylene Forming Enzyme (EFE). For more information see the <a href =" https://2013.igem.org/Team:UNITN-Trento/Safety"> <b>Safety Page. </b> </a><br />
</p><br />
<p><br />
As ripening inhibitor, we went for methyl salicylate (MeSA): an ester also known as wintergreen oil and naturally produced by many plants as a defense mechanism. It was previously shown to slow down the ripening process in tomatoes, at high concentration (5 mM) <span class="tn-ref">(Chang-Kui Ding et Al., Food Chemistry 2001, 76 213–218)</span>. To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.<br />
</p><br />
<br />
<span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q1">Do you know how plants produce ethylene?</a></span><br />
<span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q2">Do you know that ethylene is used commercially to ripen some fruits before they enter the market?</a></span><br />
<span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q3">Do you know that fruit is generally classified in two main categories?</a></span><br />
<br />
<span class="tn-subtitle">How is <i>B. fruity </i> activated?</span><br />
<p><br />
We envisioned a system that is coupled to a blue light photoreceptor, the same receptor that has been used previously by other labs and iGEM teams. Our system in the OFF state (no blue light) will produce methyl salicylate and, in the absence of ethylene, will stop unwanted ripening (<b>Figure 2</b>), while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening (<b>Figure 1</b>).<br />
</p><br />
<p><br />
Thus far we have successfully built the device shown in <b>Figure 1</b> plus many other functional genetic constructs that you can find in the <a href="https://2013.igem.org/Team:UNITN-Trento/Parts">Parts page</a>.<br />
</p><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/5/5b/Tn-2013_intro_Efe_lineare.jpg"><br />
<span class="tn-caption"><b>Fig. 1:</b> a schematic representation of ethylene production regulated by a photo-inducible circuit. The inverter ensures that in presence of blue Light 2-Oxoglutarate Oxygenase/Decarboxylase (EFE) gene can be expressed.</span><br />
<br />
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_intro_Mesa_lineare.jpg"><br />
<span class="tn-caption"><b>Fig. 2:</b> a schematic representation of methyl salicylate production regulated by a photo-repressible circuit. Blue light blocks the blue receptor cassette inducing activity, resulting in the repression of MeSA production.</span><br />
<br />
<p><br />
You can check our <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Datapage">DATA page</a> for a full description of the complete circuit.<br />
</p><br />
<br />
<span class="tn-subtitle">Why <i>B. fruity </i>?</span><br />
<p><br />
We engineered the full system and characterized each component of the system in <i>Escherichia coli</i>. We have also tried to demonstrate the functionality of the enzymes in <i>Bacillus subtilis</i>.<br />
</p><br />
<p><br />
In order to develop a possible commercial product it is more desirable to use a chassis able to survive without nutrients for a longer time: we thought that <i>Bacillus subtilis</i> could fit perfectly our purpose! It can make spores and be easily re-activated by removing the source of stress and adding, for example, water/nutrients. Moreover, <i>B. subtilis</i> is not a human pathogen. Although this bacterium can degrade or contaminate food, with the right precautions this chassis seems to be the best system for our project.<br />
</p><br />
<br />
<span class="tn-effect">Follow our results to discover how we successfully ripen fruit!</span><br />
<img style="width:60%;"src="https://static.igem.org/mediawiki/2013/d/d5/Tn-2013_fruit_img_intro.JPG"><br />
<br/><br />
</div><br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento"><br />
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</a><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/d/d0/Tn-2013-arr-HOME_next.png" /><br />
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</div><br />
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<br />
<a id="tn-sp-tour" href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene#tour"><br />
<img src="https://static.igem.org/mediawiki/2013/6/6a/Tn-2013-tour-F_AAA_DSC_0054.png" /><br />
<span>Continue the tour!</span><br />
</a><br />
<br />
<!--end content--><br />
</html>|<html>https://static.igem.org/mediawiki/2013/a/ab/Tn-2013-headerbg-Sfondosb.jpg</html>|<html>https://static.igem.org/mediawiki/2013/7/7e/Tn-2013-headingbg-Sfondosb_or.jpg</html>}}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Extra/Fruit_InfoTeam:UNITN-Trento/Extra/Fruit Info2013-10-15T12:29:07Z<p>Xli: </p>
<hr />
<div>{{:Team:UNITN-Trento/Templates/Default|<html><!--start content--><br />
<br />
<!--CSS--><br />
<link rel="stylesheet" href="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/CSS/Project_pages&amp;action=raw&amp;ctype=text/css" type="text/css"><br />
<link rel="stylesheet" href="https://2013.igem.org/Team:UNITN-Trento/CSS/Extra/Fruit_Info?action=raw&amp;ctype=text/css" type="text/css"><br />
<br />
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<script src="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/JS/Project_pages&amp;action=raw&amp;ctype=text/javascript" type="text/javascript"></script><br />
<br />
<!--PAGE--><br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Fruit Info</span><br />
<br />
<span class="tn-subtitle">Do you know that fruit is generally classified in two main categories?</span><br />
<p><br />
Fruit is generally classified in two main groups: climacteric and non-climacteric.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Climacteric fruit:</span><br />
<p><br />
Some examples are: apples, bananas, pears, melons, apricoats and tomatoes.<br><br />
This kind of fruit presents a characteristic climacteric peak, which consists in an acceleration of both cellular respiration and ethylene production. Depending on the fruit, ethylene can influence all the different steps of the ripening process like colour, pulp texture, flavour... Exogenous ethylene treatment can have an effect on these fruit before the climacteric peak.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Non-climacteric:</span><br />
<p><br />
Some examples are: strawberries, citrus, oranges, cherries and grapes.<br><br />
This type of fruit have a constant low endogenous production of ethylene with no climacteric peak. Non-climacteric fruit do not respond to exogenous ethylene treatment except for in term of degreening and they should be picked up only when completely ripened in order to mantain all the flavour quality <span class="tn-ref">(Mary Lu Arpaia et Al., Fruit Ripening &amp; Ethylene Management 2010, 3-10)</span>.<br />
</p><br />
<br />
<div id="jms-slideshow" class="jms-slideshow" style="height:250px !important;"><br />
<div id="step-1" style="height:390px !important;" class="step" data-x="0"><br />
<img style="width:100% !important;" src="https://static.igem.org/mediawiki/2013/3/38/Climacteric.jpg"><br />
</div><br />
<div id="step-2" style="height:390px !important;" class="step" data-x="500"><br />
<img style="width:100% !important;" src="https://static.igem.org/mediawiki/2013/5/51/Tn-2013Non_climacteric.jpg"><br />
</div><br />
</div><br />
<br />
<span class="tn-subtitle">Do you know how plants produce ethylene?</span><br />
<img class="no-border no-bottom" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg" /><br />
<p><br />
Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed in 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, the final enzyme of the pathway was identified: ACC oxidase, which converts ACC to ethylene.<br />
</p><br />
<br />
<span class="tn-subtitle">Do you know that ethylene is used commercially to ripen some fruits before they enter the market?</span><br />
<p><br />
All fruit, 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, and treated with ethylene during transportation or once arrived at destination, before being brought to the market. Bananas for example are threated with 10 to 200 ppm of ethylene to trigger the ripening process. This approach allows long term storage of fruit and helps the distributors to bring fruit to the consumer.<br />
</p><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Attributions"><br />
<img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/6/6a/Tn-2013-arr-FRINFO_prev.png" /><br />
</a><br />
<a href="javascript:toTop('#tn-main-wrap-wrap');"><br />
<img src="https://static.igem.org/mediawiki/2013/6/6e/Tn-2013-arr-AAA_TOP.png" /><br />
</a><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Human_Practice"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/4/44/Tn-2013-arr-FRINFO_next.png" /><br />
</a><br />
</div><br />
</div><br />
<br />
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<hr />
<div></div>Xlihttp://2013.igem.org/File:Tn-2013-mod-Interval-total.pngFile:Tn-2013-mod-Interval-total.png2013-10-15T12:27:31Z<p>Xli: uploaded a new version of &quot;File:Tn-2013-mod-Interval-total.png&quot;</p>
<hr />
<div></div>Xlihttp://2013.igem.org/File:Tn-2013-mod-Interval-total.pngFile:Tn-2013-mod-Interval-total.png2013-10-15T12:26:40Z<p>Xli: uploaded a new version of &quot;File:Tn-2013-mod-Interval-total.png&quot;</p>
<hr />
<div></div>Xlihttp://2013.igem.org/File:Tn-2013-project_ethylene-BBa_K1065000.jpgFile:Tn-2013-project ethylene-BBa K1065000.jpg2013-10-15T12:04:15Z<p>Xli: uploaded a new version of &quot;File:Tn-2013-project ethylene-BBa K1065000.jpg&quot;</p>
<hr />
<div></div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/Extra/SafetyTeam:UNITN-Trento/CSS/Extra/Safety2013-10-04T20:52:11Z<p>Xli: </p>
<hr />
<div>#tn-central-container #tn-content-wrap #tn-content .container {<br />
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}<br />
<br />
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<br />
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}<br />
<br />
.tn-double-wrap .text {<br />
display: inline-block;<br />
margin-left: 45px;<br />
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<br />
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/*Navigation arrows*/<br />
<br />
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<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-prev,<br />
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<br />
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}<br />
<br />
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position: absolute;<br />
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<br />
color: black;<br />
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<br />
#tn-sp-tour:hover span {<br />
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<br />
@media(max-width:1500px) {<br />
#tn-central-container {<br />
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}<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:link, #tn-central-container #tn-content-wrap #tn-content .container .sheet a:visited {<br />
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}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/ProtocolsTeam:UNITN-Trento/CSS/Protocols2013-10-04T20:50:33Z<p>Xli: </p>
<hr />
<div>#tn-content .container {<br />
position: static;<br />
<br />
margin: 2em auto;<br />
padding: 0;<br />
width: 960px;<br />
<br />
background-color: rgb(32,86,54);<br />
background-image: url('https://static.igem.org/mediawiki/2013/9/91/Tn-2013-boxbg-Onda-Watermelon.png');<br />
background-position: top center;<br />
background-repeat: no-repeat;<br />
border: 3px solid white;<br />
box-shadow: 2px 2px 4px #323232;<br />
<br />
text-align:justify;<br />
}<br />
<br />
#tn-content .container .sheet {<br />
margin: 2em;<br />
padding: 1em;<br />
<br />
background: rgba(255,255,255,0.7);<br />
border-radius: 1em;<br />
<br />
text-align: justify;<br />
}<br />
<br />
/*HEADER IN APPOSITE SUBS/TEMPLATE*/<br />
<br />
/*******************/<br />
/*CENTRAL CONTAINER*/<br />
/*******************/<br />
<br />
#tn-central-container {<br />
display: block;<br />
<br />
/*max-width: 1300px;*/<br />
padding-top: 2.3em !important;<br />
width: 100%;<br />
min-height: 600px;<br />
<br />
margin: 0 auto;<br />
padding: 1em 0;<br />
<br />
background-color: #dadada;<br />
<br />
box-shadow: 0 0 8px ;<br />
<br />
font-family: 'Cabin', sans-serif;<br />
}<br />
<br />
#tn-content-wrap {<br />
display: static;<br />
<br />
max-width: 1200px;<br />
min-height: 600px;<br />
<br />
margin: 0 auto;<br />
margin-top: 6em;<br />
padding: 1px;<br />
padding-top: 0;<br />
}<br />
<br />
#tn-content {<br />
display: static;<br />
<br />
max-width: 1200px;<br />
height: 100%;<br />
<br />
margin-top: -0.8em;<br />
padding: 1em;<br />
padding-top: 0;<br />
}<br />
<br />
#tn-content a {<br />
color: #323232;<br />
}<br />
<br />
#tn-content a:hover {<br />
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}<br />
<br />
.tn-spoiler-protocol > a {<br />
display: block;<br />
<br />
padding: 0.2em;<br />
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<br />
background: #F0F0F0;<br />
border: 1px solid #2880C5;<br />
box-shadow: 1px 1px 1px #323232;<br />
color: #2880C5!important;<br />
<br />
font-variant: small-caps;<br />
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<br />
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border: 1px solid #DCD8D0;<br />
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<br />
.tn-spoiler-protocol div {<br />
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padding: 10px;<br />
text-align: justify;<br />
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}<br />
<br />
.tn-spoiler-protocol div table {<br />
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<br />
.tn-spoiler-protocol div th {<br />
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background-color: #bcbcbc;<br />
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<br />
table.results td {<br />
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<br />
.tn-spoiler-protocol > div {<br />
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<br />
background: #f0f0f0;<br />
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<br />
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<br />
#tn-content h2 {<br />
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border-bottom: 2px solid #2880C5;<br />
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<br />
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font-style: normal;<br />
<br />
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text-align: left;<br />
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<br />
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<br />
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<br />
text-align: center;<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-prev,<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-next {<br />
position: absolute;<br />
bottom: 0.8em;<br />
<br />
width: 174px;<br />
<br />
border: none;<br />
box-shadow: none;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-prev {<br />
left: 2em;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-next {<br />
right: 2em;<br />
}<br />
<br />
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color:rgb(32,86,54);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:hover {<br />
color:black; <br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:link, #tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:visited {<br />
color:rgb(32,86,54);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/Project/ModelingTeam:UNITN-Trento/CSS/Project/Modeling2013-10-04T20:48:00Z<p>Xli: </p>
<hr />
<div>#tn-content .container {<br />
background-color: rgb(253,209,26);<br />
background-image: url(https://static.igem.org/mediawiki/2013/4/4f/Tn-2013-boxbg-Onda-Lemon.png);<br />
}<br />
<br />
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<br />
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<br />
text-align: center;<br />
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<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-prev,<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-next {<br />
position: absolute;<br />
bottom: 0.8em;<br />
<br />
width: 174px;<br />
<br />
border: none;<br />
box-shadow: none;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-prev {<br />
left: 2em;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-next {<br />
right: 2em;<br />
}<br />
<br />
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color:rgb(245, 90, 0);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:hover {<br />
color:black; <br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:link, #tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:visited {<br />
color:rgb(245, 90, 0);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/Project/ModelingTeam:UNITN-Trento/CSS/Project/Modeling2013-10-04T20:46:47Z<p>Xli: </p>
<hr />
<div>#tn-content .container {<br />
background-color: rgb(253,209,26);<br />
background-image: url(https://static.igem.org/mediawiki/2013/4/4f/Tn-2013-boxbg-Onda-Lemon.png);<br />
}<br />
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<br />
margin: -1em 0 1em 0;<br />
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#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-prev,<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-next {<br />
position: absolute;<br />
bottom: 0.8em;<br />
<br />
width: 174px;<br />
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border: none;<br />
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}<br />
<br />
.container .sheet-2 .tn-arr-prev {<br />
left: 2em;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-next {<br />
right: 2em;<br />
}<br />
<br />
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color:rgb(128, 123, 0);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:hover {<br />
color:black; <br />
}<br />
<br />
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color:rgb(128, 123, 0);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/Project/ModelingTeam:UNITN-Trento/CSS/Project/Modeling2013-10-04T20:30:49Z<p>Xli: </p>
<hr />
<div>#tn-content .container {<br />
background-color: rgb(253,209,26);<br />
background-image: url(https://static.igem.org/mediawiki/2013/4/4f/Tn-2013-boxbg-Onda-Lemon.png);<br />
}<br />
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position: relative;<br />
<br />
margin: -1em 0 1em 0;<br />
padding: 0.5em;<br />
<br />
background-color: rgba(255,255,255,.6);<br />
<br />
text-align: center;<br />
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#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-next {<br />
position: absolute;<br />
bottom: 0.8em;<br />
<br />
width: 174px;<br />
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border: none;<br />
box-shadow: none;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-prev {<br />
left: 2em;<br />
}<br />
<br />
.container .sheet-2 .tn-arr-next {<br />
right: 2em;<br />
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<br />
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}<br />
<br />
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color:black; <br />
}<br />
<br />
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color:rgb(187, 180, 0);<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/CSS/Project/ModelingTeam:UNITN-Trento/CSS/Project/Modeling2013-10-04T20:30:23Z<p>Xli: </p>
<hr />
<div>#tn-content .container {<br />
background-color: rgb(253,209,26);<br />
background-image: url(https://static.igem.org/mediawiki/2013/4/4f/Tn-2013-boxbg-Onda-Lemon.png);<br />
}<br />
<br />
/*Navigation arrows*/<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 {<br />
position: relative;<br />
<br />
margin: -1em 0 1em 0;<br />
padding: 0.5em;<br />
<br />
background-color: rgba(255,255,255,.6);<br />
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#tn-central-container #tn-content-wrap #tn-content .container .sheet-2 .tn-arr-prev,<br />
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color:rgb(187, 180, 0));<br />
}<br />
<br />
#tn-central-container #tn-content-wrap #tn-content .container .sheet a:hover {<br />
color:black; <br />
}<br />
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#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:link, #tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:visited {<br />
color:rgb(187, 180, 0));<br />
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#tn-central-container #tn-content-wrap #tn-content .container .sheet .tn-effect-link a:hover {<br />
color:black;<br />
}</div>Xlihttp://2013.igem.org/File:Tn-2013-mod-Dark_modeling.jpgFile:Tn-2013-mod-Dark modeling.jpg2013-10-04T17:45:29Z<p>Xli: uploaded a new version of &quot;File:Tn-2013-mod-Dark modeling.jpg&quot;</p>
<hr />
<div></div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/Methyl_SalicylateTeam:UNITN-Trento/Project/Methyl Salicylate2013-10-04T14:26:48Z<p>Xli: </p>
<hr />
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<div class="sheet"><br />
<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here. </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<br />
</div><br />
<center><img class="plot"style="width:49%;border: 3px solid white;<br />
box-shadow: 2px 2px 4px #323232;margin-top: -6px;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA (data not shown).<br />
</p><br/><br />
<span class="tn-title">Summary</span><br />
Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br><br><br />
After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
</div><br />
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<!--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>}}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/Methyl_SalicylateTeam:UNITN-Trento/Project/Methyl Salicylate2013-10-04T14:25:01Z<p>Xli: </p>
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<div class="sheet"><br />
<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here. </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<br />
</div><br />
<center><img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA (data not shown).<br />
</p><br/><br />
<span class="tn-title">Summary</span><br />
Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br><br><br />
After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
</div><br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene"><br />
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<!--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>}}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/Methyl_SalicylateTeam:UNITN-Trento/Project/Methyl Salicylate2013-10-04T12:09:36Z<p>Xli: </p>
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<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA.<br />
</p><br/><br />
<span class="tn-title">Summary</span><br />
Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
</div><br />
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<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) standard curve corresponding to 15 ppm.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salycilici acid). Non induced cells did not produce any MeSA.<br />
</p><br/><br />
<span class="tn-title">Summary</span><br />
Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
</div><br />
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<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) standard curve corresponding to 15 ppm.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salycilici acid). Non induced cells did not produce any MeSA.<br />
</p><br />
<span class="tn-title">Summary</span><br />
Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
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<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img style="width:32%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img style="width:32%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<img style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) standard curve corresponding to 15 ppm.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salycilici acid). Non induced cells did not produce any MeSA.<br />
</p><br />
<span class="tn-title">Summary</span><br />
Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
<br />
</div><br />
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<!--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>}}</div>Xlihttp://2013.igem.org/Team:UNITN-Trento/Project/Methyl_SalicylateTeam:UNITN-Trento/Project/Methyl Salicylate2013-10-04T11:56:49Z<p>Xli: </p>
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<span class="tn-title">Results - Methyl Salicylate </span><br />
<p> <br />
<i>B. fruity</i> needed also a fruit ripening ihnibitor. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also proofed to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous work suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. <br />
</p><br />
<p><br />
We were happy to find out that many of the needed parts to produce MeSA were already available in the registry. These parts were initally built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.<br />
</p><br />
<br />
<img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/><br />
<br />
<span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism <i>Pseudomonas aeruginosa </i>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in the genome of <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an increase of MeSA production. </span><br />
<br />
<p><br />
We modified and improved these parts and resubmitted them to the registry. For example, we substituted the pTet promoter controlling the BSMT1 enzyme with an araC-pBAD promoter. Additionally the MIT team did not include in their MeSA generator device the enzyme SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 the single enzymes of the pathway.<br />
</p><br />
<br />
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg" /><br />
<br />
<span class="tn-subtitle">MeSA detection</span> <br />
<p><br />
MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> <br />
</p><br />
<br />
<div class= "tn-doublephoto-wrap"><br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg"/> <br />
<img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /><br />
</div><br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 3:</b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some problems with the solubility of the methyl salicylate. However, during our experience were also performed many measures to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span><br />
<br />
Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium, induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography measures here reported were done in liquid phase, by injecting 1 ul of pre-filtered culture in the instrument.<br />
<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"><br />
<span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue trace) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red trace).</span><br />
<br />
Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each bacteria’s samples. Below we have reported the most significant data.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/><br />
<br />
<span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> Quantification of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span><br />
<span class="tn-subtitle">MeSA: 1ppm is better than zero</span><br />
<p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p><br />
<div class="tn-doublephoto-wrap"><br />
<img src="https://2013.igem.org/File:Tn-2013_BBa_K1065102_induced.jpg"/><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/><br />
<img src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) standard curve corresponding to 15 ppm.</span><br />
<p><br />
NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salycilici acid). Non induced cells did not produce any MeSA.<br />
</p><br />
<span class="tn-title">Summary</span><br />
Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.<br />
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