http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Cridelbianco&year=&month=2013.igem.org - User contributions [en]2024-03-29T10:10:17ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UNITN-Trento/Project/EthyleneTeam:UNITN-Trento/Project/Ethylene2013-10-28T22:13:53Z<p>Cridelbianco: </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, 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 (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. 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 />
<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; 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 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, 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 (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. 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 />
<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; 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 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, 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 (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">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our device was designed with a photoinducible system in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in manufacturing and ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine is designed to have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our device was designed with a photoinducible system in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in manufacturing and ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our device was designed with a photoinducible system in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in manufacturing or ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><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 />
</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"><br />
Bacillus subtilis<br />
</span><br />
When we first came up with the idea of <i>B. fruity</i>, we immediatly thought that <i>B. subtilis</i> was the perfect chassis for a possible marketable application:<br />
<ol><br />
<li><br />
<i>Bacillus subtilis</i> sporulates and it can be stored in a inactive state;<br />
</li> <br />
<li><br />
<i>Bacillus subtilis</i> is not pathogenic and therefore can be used safely for food applications; <br />
</li><br />
<br />
</ol><br />
<br />
<i>Bacillus subtilis</i> would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a <i>B. fruity</i> home edition that exploits this principle. <br/><br />
To achieve this goal we started working with EFE, a ethylene forming enzyme from <i>Pseudomas Syringae</i> pv. phaseolicola (<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>), which were inserted into pSBBs0K-Pspac (IPGT inducible) and pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for <i>B. subtilis</i> by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were kindly sent to us from LMU Munich).<br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/><br />
<br />
<span class="tn-subtitle">Cloning of BBa_K1065204</span><br />
The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into <i>B. subtilis</i> genome was confirmed with the threonine assay.<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b0/Tn-2013_PXyl_digestion.png"/><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/3/3b/Tn-2013_thr_assay.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 1:</b> transformation of <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_1065204</a> in <i>B. subtilis</i>. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into <i>B. subtilis</i> 168 using minimal medium. Correct integration was confirmed with the threonine test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine.</span><br />
<br />
<span class="tn-subtitle">Toxicity assay</span><br />
We then measured the optical density of cells induced and non induced for both constructs.<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/1/14/Tn-2013_K1065204_plot.png"> <br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/8/8e/Tn-2013_K1065203_plot.png"> <br />
</div><br />
<span class="tn-caption"><b>Figure 2:</b> <i>B. subtilis</i> 168 cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> or <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> were grown until an OD=0.9 and then splitted in two samples before induction. Cells were induced with 1% xylose for BBa_K1065204 and 0.5 mM of IPTG for BBa_K1065203. In both cases the induced samples (blue trace) grow slightly slower than the controls (red trace).</span><br />
<span class="tn-subtitle">Sporulation assay</span><br />
Spores were obtained by growing the transformed <i>B. subtilis</i> 168 cells in DSM medium, subjecting them to a heat shock at 60 &deg;C and plating them on a preheated glass slide. Spores were visualized at the microscope.<br />
<span class="tn-subtitle">Ethylene detection</span><br />
Ethylene production was tested by Gas Chromatography as we previoulsy did for <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>. The experiment was performed both from cultures started from fresh plates and from dry spores.<br/><br />
We did not observe any production of ethylene after 4 hours, nor after overnight induction. <br/><br />
At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip. <br />
<img src="https://static.igem.org/mediawiki/2013/6/69/Tn-2013_Lead-acetate_strip_assay.jpg"/><br />
<span style="text-align:justify;"class="tn-caption center"><b>Figure 4:</b> detection of sulfur compounds. <i>B. subtilis</i> 168 cells non transformed (1 and 2), transformed with <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> and transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1% xylose or with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate. </span><br />
<span class="tn-subtitle">Future directions</span><br />
For future experiments and improvement of the system we have identified additional potential drawbacks, including:<br />
<ul><br />
<li>pXyl could be inhibited by glucose although the threonine test confirmed the correct insertion of the vector;</li><br />
<li>the acquisition of pSpac could not be confirmed by colony PCR yet; even if the growth of colonies in the presence of the antibiotic indicates that the episomal vector carrying EFE gene is present;</li><br />
<li>at present the expression of the EFE gene has not been demonstrated. We plan to perform an additional real-time PCR experiment (to assess transcription).</li><br />
</ul> <br />
</div><br />
<br />
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<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our device was designed with a photoinducible system in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our system was designed to be inducible in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our system was designed to be inducible in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, our vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<br />
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<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our system was designed to be inducible in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
</div><br />
<br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a safer alternative pathway.</li><br />
<li>our system was designed to be inducible in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
</div><br />
<br />
<div class="sheet-2"><br />
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<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the more commonly studied plant ethylene pathway, because this plant pathway also produces cyanide. Therefore, we selected a more safe alternative pathway.</li><br />
<li>our system was designed to be inducible in order to avoid the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene for ripening facilities.</li><br />
</ul><br />
For more detailed information please read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we discovered a problem: the plant pathway produces hydrogen cyanide as a byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>P. syringae</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example ethylene exposure to employees is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the plant ethylene pathway, which also produces cyanide, and we selected a more safe alternative pathway.</li><br />
<li>our system was designed to be inducible and to avoid accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities.</li><br />
</ul><br />
For more detailed information read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example employees ethylene exposure is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group, BBa_J45319 that catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
</div><br />
<br />
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<br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the plant ethylene pathway, which also produces cyanide, and we selected a more safe alternative pathway.</li><br />
<li>our system was designed to be inducible and to avoid accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities.</li><br />
</ul><br />
For more detailed information read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example employees ethylene exposure is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk if ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<div class="sheet"><br />
<span class="tn-title">Safety considerations</span><br />
<p><br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<ul><br />
<li>we decided not to use the plant ethylene pathway, which also produces cyanide, and we selected a more safe alternative pathway.</li><br />
<li>our system was designed to be inducible and to avoid accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities.</li><br />
</ul><br />
For more detailed information read the safety considerations below.<br /><br />
</p><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins. <font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br /><br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br /><br />
<br /><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br /><br />
<br /><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. Ethylene handling requires many precautions, for example employees ethylene exposure is highly controlled in manufacturing facilities.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year many accidents are reported that are caused by the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)(<a href="http://www.factsonline.nl/accidents/%205405/91038_ETHYLENE%20%28COOLED%29/chemical-accidents-with-ethylene-%28cooled%29/">FACTS - Hazardous Material Accidents Knowledge Base</a>)</span><br />
</p><br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold.<br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
</p><br />
<p><br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<span class="tn-title">Human Practice</span><br />
<br />
<span class="subtitle">A very fruity survey!</span><br />
<p><br />
After we got the idea of a <i>B. fruity</i> vending machine, we wanted to verify the opinion of local people on our concept. <br />
</p><br />
<p><br />
We proposed a five-questions survey to fruit consumers in the local market.<br />
<ol><br />
<li><br />
What do you think about a vending machine selling fruit? Could it be useful to consume more fruit?<br />
</li><br />
<li><br />
Do you agree on using 2 molecules (Ethylene and Methyl salicylate) either to speed up or slow down fruit ripening?<br />
</li><br />
<li><br />
Did you know that bananas are imported completely green and exposed to ethylene to make them ready-to-eat before they are presented on the market?<br />
</li><br />
<li><br />
What if ethylene is produced by harmless bacteria that do not come in contact with fruit: would you still eat fruit exposed to ethylene?<br />
</li><br />
<li><br />
Do you think that bacterial ethylene somehow differs from the plant ethylene or the chemically synthesized one?<br />
</li><br />
</ol><br />
</p><br />
<p><br />
Then, one Thursday of August the team went downtown at the city market. There we tested the knowledge level of the community on fruit ripening and asked if they thought that a fruit vending machine might be useful.<br />
</p><br />
<img style="width: 600px;" src="https://static.igem.org/mediawiki/2013/c/ce/Tn-2013-HPsurv1-556861_505452562869419_996488170_n.jpg" /><br />
<p><br />
Also an online form was prepared (powered by GoogleDrive Forms) to obtain more results in a more standard way.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/Tn-2013-HPsurv2-1236010_505452532869422_2003614255_n.jpg" /><br />
<p><br />
Here are the results of our analysis.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Market survey</span><br />
<div class="jms-slideshow" id="jms-market-slideshow"><br />
<div id="graph1" class="step"><br />
<span class="q">1. What do you think about a vending machine selling fruit? Could it be useful to consume more fruit?</span><br />
<img src="https://static.igem.org/mediawiki/2013/c/c9/Tn-2013-market-question1.png" alt="" class="left"><br />
<div class="right"><br />
The first question had generally favorable results indicating the good impact of our vending machine on the surveyed sample.<br />
</div><br />
</div><br />
<br />
<div id="graph2" class="step"><br />
<span class="q">2. Do you agree on using 2 molecules (Ethylene and Methyl salicylate) either to speed up or slow down fruit ripening?</span><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/Tn-2013-market-question2.png" alt="" class="left"><br />
<div class="right"><br />
Difference between answers is very small, it seems that the surveyed people might as well do or do not agree with the ethylene/MeSA fruit treatment.<br />
</div><br />
</div><br />
<br />
<div id="graph3" class="step"><br />
<span class="q">3. Did you know that bananas are imported completely green and exposed to ethylene to make them ready-to-eat before they are presented on the market?</span><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/Tn-2013-market-question3.png" alt="" class="left"><br />
<div class="right"><br />
The answers difference is very small in this question too. It seems that presence or absence of prior knowledge in regards of bananas ethylene treatment are equally probable.<br />
</div><br />
</div><br />
<br />
<div id="graph4" class="step"><br />
<span class="q">4. What if ethylene is produced by harmless bacteria that do not come in contact with fruit: would you still eat fruit exposed to ethylene?</span><br />
<img src="https://static.igem.org/mediawiki/2013/8/83/Tn-2013-market-question4.png" alt="" class="left"><br />
<div class="right"><br />
Surprisingly the majority of the tested people agree with the use of ethylene producing bacteria to treat fruit.<br />
</div><br />
</div><br />
<br />
<div id="graph5" class="step"><br />
<span class="q">5. Do you think that bacterial ethylene somehow differs from the plant ethylene or the chemically synthesized one?</span><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/Tn-2013-market-question5.png" alt="" class="left"><br />
<div class="right"><br />
From the answers it seems that tested people might equally distribute between those that give a correct answer, a wrong answer or that do not have any opinion on the subject. The last two groups are characterized by a lack of prior knowledge on the subject.<br />
</div><br />
</div><br />
</div><br />
<br />
<span class="tn-sub-subtitle">Online survey</span><br />
<div class="jms-slideshow" id="jms-online-slideshow"><br />
<div id="graph1" class="step"><br />
<span class="q">1. What do you think about a vending machine selling fruit? Could it be useful to consume more fruit?</span><br />
<img src="https://static.igem.org/mediawiki/2013/a/ae/Tn-2013-online-question1.png" alt="" class="left"><br />
<div class="right"><br />
Results of the online survey is similar to the market survey as far as the first question is concerned.<br />
</div><br />
</div><br />
<br />
<div id="graph2" class="step"><br />
<span class="q">2. Do you agree on using 2 molecules (Ethylene and Methyl salicylate) either to speed up or slow down fruit ripening?</span><br />
<img src="https://static.igem.org/mediawiki/2013/e/eb/Tn-2013-online-question2.png" alt="" class="left"><br />
<div class="right"><br />
It seems that the use of molecules to control fruit ripening would be accepted by the majority of the tested people. Nearly fifty percent of the sample worked or studied on this field, so it should not be interfering, but we cannot strongly affirm this.<br />
</div><br />
</div><br />
<br />
<div id="graph3" class="step"><br />
<span class="q">3. Did you know that bananas are imported completely green and exposed to ethylene to make them ready-to-eat before they are presented on the market?</span><br />
<img src="https://static.igem.org/mediawiki/2013/9/93/Tn-2013-online-question3.png" alt="" class="left"><br />
<div class="right"><br />
Tested people might as well do or do not know that bananas are treated with ethylene.<br />
</div><br />
</div><br />
<br />
<div id="graph4" class="step"><br />
<span class="q">4. What if ethylene is produced by harmless bacteria that do not come in contact with fruit: would you still eat fruit exposed to ethylene?</span><br />
<img src="https://static.igem.org/mediawiki/2013/7/7f/Tn-2013-online-question4.png" alt="" class="left"><br />
<div class="right"><br />
Surprisingly the majority of the tested sample agreed with the usage of ethylene producing bacteria to treat fruit.<br />
</div><br />
</div><br />
<br />
<div id="graph5" class="step"><br />
<span class="q">5. Do you think that bacterial ethylene somehow differs from the plant ethylene or the chemically synthesized one?</span><br />
<img src="https://static.igem.org/mediawiki/2013/d/d6/Tn-2013-online-question5.png" alt="" class="left"><br />
<div class="right"><br />
From the response we deduced the tested people have the knowledge required to give a correct answer to this question.<br />
</div><br />
</div><br />
</div><br />
<br />
<span class="tn-sub-subtitle">Analysis</span><br />
<p><br />
We planned to perform a statistical analysis of the data, to infer the relationships between the answers and different categories like age, instruction, gender,... but then we realized that both samples, ‘market’ and ‘online’, are not representative of the population that we wanted to analyze (italian population). Also the size of the sample is not high enough: 102 from the market and 172 from the online survey.<br />
</p><br />
<br />
<span class="tn-sub-subtitle">Conclusion</span><br />
<p><br />
In conclusion, from our survey it seems that the idea of a vending machine with ready-to-eat fruit treated with bacteria-derived ethylene should have a great feedback when presented on the market!<br />
</p><br />
<img style="width: 600px;" src="https://static.igem.org/mediawiki/2013/6/62/Tn-2013-HPsurv3-533755_505452466202762_1252000404_n.jpg" /><br />
<br />
<div class="col one"><br />
<span class="subtitle">Margherita: Future iGEMers grow up </span> <br />
During the summer we had the pleasure to host for two weeks Margherita, an enthusiastic student from a High School in Trento where she is going to attend her fourth year. We introduced her to the wonders of synthetic biology and she completely understood the spirit of the iGEM competition. In particular, in these two intense weeks that we spent together, she had the possibility to see how IGEMers life is and to discover the world of biology. Day by day she discovered her passion for synthetic biology and, after some lessons, she was able to design and build her fist biobrick: pLac with amilCP. Unfortunately we discovered too late that the amilCP part that we used (<a href="http://parts.igem.org/Part:BBa_K592009"></a>BBa_K592009) didn't have a ribosomal binding site. Moreover, we discussed with her about some parts of our project and she was able to give precious advices on the project with her different point of view. In conclusion it was a great experience and a great opportunity for both the team and Margherita. A few weeks later we received a beautiful present from her: she made our logo with playdoh!<br />
</div> <br />
<div id="jms-slideshow" class="jms-slideshow col two"><br />
<div id="im1" class="step"><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013_margherita_1-resized.jpg"></a><br />
</div><br />
<div id="im2" class="step"><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013_margherita_2.jpg"></a><br />
</div><br />
<div id="im3" class="step"><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/c/c3/Tn-2013_margherita_3.jpg"></a><br />
</div><br />
<div id="im4" class="step"><br />
<a href=""><img src="https://static.igem.org/mediawiki/2013/6/66/Tn-2013_margherita_4.jpg"></a><br />
</div><br />
</div><br />
<br />
<span class="tn-subtitle">One day with Saverio Tommasi</span><br />
<p><br />
We had the pleasure to entertain in our lab for one day <a href="http://www.saveriotommasi.it/biografia/">Saverio Tommasi</a>: a giornalist, blogger, opinionist. Saverio contacted us through facebook because he was interested in documenting the life of young scientists in Italy. He observed and filmed with great interest our work and decided to tell the story of our project and the spirit of the iGEM competition in a reportage which will be released soon on the blog <a href="http://youmedia.fanpage.it/user/SaverioTommasi">fanpage.it</a>. We explained to Saverio the importance of Synthetic Biology and scientific research in general and we hope that this video will help people to approach the beautiful world of science. <br />
</p> <br />
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<span class="tn-title"><i>B. fruity</i> - Home Edition<span class="tn-subscript" style="padding-left:0!important;">Our household theoretical product.</span></span><br />
<span class="tn-subtitle">Why?</span><br />
<p><br />
Home Edition is conceived thinking about what families and regular fruit consumer need: a further way to control fruit ripening <b>at home</b>. There are plenty of occasions in which getting ripe fruit in time is needed, and just as much in which we want our fruit to stay green as long as possible. Home Edition is a reliable, easy to use, paper bag that can speed up or slow down ripening of the fruit that you can find in any family’s kitchen!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_Home_edition_1.jpg" /><br />
<p><br />
We chose paper as the main material since fruit perspiration would be restricted if the bag was in plastic. The sack is designed to have a little container on the inside called <b>the pocket</b>: it contains two semi-permeable pods with engineered <i>B. subtilis</i> that allow the molecules (in one case ethylene and in the other MeSA) to exit and act on fruit, but at the same time avoid Bacillus to get outside. We went for <i>Bacillus subtilis</i> as our chassis because it forms endospores, so it can be stored in a dormant and safe state. Taking in consideration the marketability of the idea, the capacity to turn into spores seems to be convenient and suitable for Home Edition.<br><br />
<br />
</p><br />
<span class="tn-subtitle">The pocket</span><br />
<p><br />
The paper bag is equipped with two different plastic pods that can be placed in the pocket allowing whether ethylene production or MeSA production: both are made of two compartments, one with bacillus spores, and the other (breakable) with nutrient and the respective inducer. <br />
The pod is built with a plastic material that allows ethylene/MeSA to escape in the paper bag, but entraps the bacteria inside the pod itself.<br />
For this device we took inspiration from groeningen 2012 igem team’s “sticker”, a fascinating device that they designed and actually built in order to detect and report volatiles in spoiling meat.<br />
After breaking the partition wall between the two chambers, the nutrition goes to the spores reactivating them; the inducer enables the bacteria to produce the molecule demanded, that will go out through this special semi-permeable plastic. <br />
Therefore ethylene/ MeSA production is avoided until nutrients and inducer are added.<br />
The two different sachets are coloured differently, for an easy-to-use application, one is blue-colored (MeSA) and the other is orange (ethylene).<br />
</p><br />
<br />
<span class="tn-effect">Now you have to choose which color you want to pop!!</span><br />
<br />
<p><br />
Once you break the desired compartment, you just put the pod inside the pocket then zip the bag and here we go!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013_Home_edition_2.jpg" /><br />
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<span class="tn-title"><i>B. fruity</i> - Home Edition<span class="tn-subscript" style="padding-left:0!important;">Our household theoretical product.</span></span><br />
<span class="tn-subtitle">Why?</span><br />
<p><br />
Home Edition is conceived thinking about what families and regular fruit consumer need: a further way to control fruit ripening <b>at home</b>. There are plenty of occasions in which getting ripe fruit in time is needed, and just as much in which we want our fruit to stay green as long as possible. Home Edition is a reliable, easy to use, paper bag that can speed up or slow down ripening of the fruit that you can find in any family’s kitchen!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_Home_edition_1.jpg" /><br />
<p><br />
We chose paper as the main material since fruit perspiration would be restricted if the bag was in plastic. The sack is designed to have a little container on the inside called <b>the pocket</b>: it contains two semi-permeable pods with engineered <i>B. subtilis</i> that allow the molecules (in one case ethylene and in the other MeSA) to exit and act on fruit, but at the same time avoid Bacillus to get outside. We went for <i>Bacillus subtilis</i> as our chassis because it forms endospores, so it can be stored in a dormant and safe state. Taking in consideration the marketability of the idea, the capacity to turn into spores seems to be convenient and suitable for Home Edition.<br><br />
<br />
</p><br />
<span class="tn-subtitle">The pocket</span><br />
<p><br />
The paper bag is equipped with two different plastic pods that can be placed in the pocket allowing whether ethylene production or MeSA production: both are made of two compartments, one with bacillus spores, and the other (breakable) with nutrient and the respective inducer. <br />
The pod is built with a plastic material that allows ethylene/MeSA to escape in the paper bag, but entraps the bacteria inside the pod itself.<br />
For this device we took inspiration from groeningen 2012 igem team’s “sticker”, a fascinating device that they designed and actually built in order to detect and report volatiles in spoiling meat.<br />
After breaking the partition wall between the two chambers, the nutrition goes to the spores reactivating them; the inducer enables the bacteria to produce the molecule demanded, that will go out through this special semi-permeable plastic. <br />
Therefore ethylene/ MeSA production is avoided until nutrients and inducer are added.<br />
The two different sachets are coloured differently, for an easy-to-use application, one is blue-colored (MeSA) and the other is orange (ethylene).<br />
</p><br />
<br />
<span class="tn-effect">Now you have to choose which color you want to pop!!</span><br />
<br />
<p><br />
Once you break the desired broth compartment, you just put the sachet inside the pocket then close the gap on the top and here we go!!<br />
</p><br />
<br />
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<span class="tn-title"><i>B. fruity</i> - Home Edition<span class="tn-subscript" style="padding-left:0!important;">Our household theoretical product.</span></span><br />
<span class="tn-subtitle">Why?</span><br />
<p><br />
Home Edition is conceived thinking about what families and regular fruit consumer need: a further way to control fruit ripening <b>at home</b>. There are plenty of occasions in which getting ripe fruit in time is needed, and just as much in which we want our fruit to stay green as long as possible. Home Edition is a reliable, easy to use, paper bag that can speed up or slow down ripening of the fruit that you can find in any family’s kitchen!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_Home_edition_1.jpg" /><br />
<p><br />
We chose paper as the main material since fruit perspiration would be restricted if the bag was in plastic. The sack is designed to have a little container on the inside called <b>the pocket</b>: it contains two semi-permeable pods with engineered <i>B. subtilis</i> that allow the molecules (in one case ethylene and in the other MeSA) to exit and act on fruit, but at the same time avoid Bacillus to get outside. We went for <i>Bacillus subtilis</i> as our chassis because it forms endospores, so it can be stored in a dormant and safe state. Taking in consideration the marketability of the idea, the capacity to turn into spores seems to be convenient and suitable for Home Edition.<br><br />
Therefore ethylene/ MeSA production is avoided until nutrients and inducer are added.<br><br />
The pod is built with a plastic material that allows ethylene/MeSA to escape in the paper bag, but entraps the bacteria inside the pod itself.<br />
For this device we took inspiration from groeningen 2012 igem team’s “sticker”, a fascinating device that they designed and actually built in order to detect and report volatiles in spoiling meat.<br />
</p><br />
<span class="tn-subtitle">The pocket</span><br />
<p><br />
The paper bag is equipped with two different plastic pods that can be placed in the pocket allowing whether ethylene production or MeSA production: both are made of two compartments, one with bacillus spores, and the other (breakable) with nutrient and the respective inducer. After breaking the partition wall between the two chambers, the nutrition goes to the spores reactivating them; the inducer present in the broth enables the culture to produce the molecule demanded, that will go out through this special semi-permeable plastic. Besides, to recognize which sachet produces ethylene and wich MeSA, one is blue-colored (MeSA) and the other is orange (ethylene).<br />
</p><br />
<br />
<span class="tn-effect">Now you have to choose which color you want to pop!!</span><br />
<br />
<p><br />
Once you break the desired broth compartment, you just put the sachet inside the pocket then close the gap on the top and here we go!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013_Home_edition_2.jpg" /><br />
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<span class="tn-title"><i>B. fruity</i> - Home Edition<span class="tn-subscript" style="padding-left:0!important;">Our household theoretical product.</span></span><br />
<span class="tn-subtitle">Why?</span><br />
<p><br />
Home Edition is conceived thinking about what families and regular fruit consumer need: a further way to control fruit ripening <b>at home</b>. There are plenty of occasions in which getting ripe fruit in time is needed, and just as much in which we want our fruit to stay green as long as possible. Home Edition is a reliable, easy to use, paper bag that can speed up or slow down ripening of the fruit that you can find in any family’s kitchen!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_Home_edition_1.jpg" /><br />
<p><br />
We chose paper as the main material since fruit perspiration would be restricted if the bag was in plastic. The sack is designed to have a little container on the inside called <b>the pocket</b>: it contains two semi-permeable pods with engineered <i>B. subtilis</i> that allow the molecules (in one case ethylene and in the other MeSA) to exit and act on fruit, but at the same time avoid Bacillus to get outside. We went for <i>Bacillus subtilis</i> as our chassis because it forms endospores, so it can be stored in a dormant and safe state. Taking in consideration the marketability of the idea, the capacity to turn into spores seems to be convenient and suitable for Home Edition.<br><br />
Therefore ethylene/ MeSA production is avoided until nutrients and inducer are added.<br><br />
For this device we took inspiration from groeningen 2012 igem team’s “sticker”, a fascinating device that they designed and actually built in order to detect and report volatiles in spoiling meat.<br />
</p><br />
<span class="tn-subtitle">The pocket</span><br />
<p><br />
The paper bag is equipped with two different plastic pods that can be placed in the pocket allowing whether ethylene production or MeSA production: both are made of two compartments, one with bacillus spores, and the other (breakable) with nutrient and the respective inducer. After breaking the partition wall between the two chambers, the nutrition goes to the spores reactivating them; the inducer present in the broth enables the culture to produce the molecule demanded, that will go out through this special semi-permeable plastic. Besides, to recognize which sachet produces ethylene and wich MeSA, one is blue-colored (MeSA) and the other is orange (ethylene).<br />
</p><br />
<br />
<span class="tn-effect">Now you have to choose which color you want to pop!!</span><br />
<br />
<p><br />
Once you break the desired broth compartment, you just put the sachet inside the pocket then close the gap on the top and here we go!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013_Home_edition_2.jpg" /><br />
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<span class="tn-title"><i>B. fruity</i> - Home Edition<span class="tn-subscript" style="padding-left:0!important;">Our household theoretical product.</span></span><br />
<span class="tn-subtitle">Why?</span><br />
<p><br />
Home Edition is conceived thinking about what families and regular fruit consumer need: a further way to control fruit ripening <b>at home</b>. There are plenty of occasions in which getting ripe fruit in time is needed, and just as much in which we want our fruit to stay green as long as possible. Home Edition is a reliable, easy to use, paper bag that can speed up or slow down ripening of the fruit that you can find in any family’s kitchen!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_Home_edition_1.jpg" /><br />
<p><br />
We chose paper as the main material since fruit perspiration would be restricted if the bag was in plastic. The sack is designed to have a little container on the inside called <b>the pocket</b>: it contains two semi-permeable pods with engineered <i>B. subtilis</i> that allow the molecules (in one case ethylene and in the other MeSA) to exit and act on fruit, but at the same time avoid Bacillus to get outside. We went for <i>Bacillus subtilis</i> as our chassis because it forms endospores, so it can be stored in a dormant and safe state. Taking in consideration the marketability of the idea, the capacity to turn into spores seems to be convenient and suitable for Home Edition.<br><br />
Therefore ethylene/ MeSA production is avoided until fresh broth is added.<br><br />
For this device we took inspiration from groeningen 2012 igem team’s “sticker”, a fascinating device that they designed and actually built in order to detect and report volatiles in spoiling meat.<br />
</p><br />
<span class="tn-subtitle">The pocket</span><br />
<p><br />
The paper bag is equipped with two different plastic pods that can be placed in the pocket allowing whether ethylene production or MeSA production: both are made of two compartments, one with bacillus spores, and the other (breakable) with nutrient and the respective inducer. After breaking the partition wall between the two chambers, the nutrition goes to the spores reactivating them; the inducer present in the broth enables the culture to produce the molecule demanded, that will go out through this special semi-permeable plastic. Besides, to recognize which sachet produces ethylene and wich MeSA, one is blue-colored (MeSA) and the other is orange (ethylene).<br />
</p><br />
<br />
<span class="tn-effect">Now you have to choose which color you want to pop!!</span><br />
<br />
<p><br />
Once you break the desired broth compartment, you just put the sachet inside the pocket then close the gap on the top and here we go!!<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013_Home_edition_2.jpg" /><br />
</div><br />
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<span class="tn-title">Judging</span><br />
<br />
<div class="bronze medal"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Tn-2013Bronze-medalaaaaa.png" /><br />
</div><br />
<div class="bronze text"><br />
<span class="tn-subtitle">Requirements for a Bronze Medal:</span><br />
<ul class="apple-list"><br />
<li><br />
<a href="https://igem.org/Team.cgi">Register the team</a>, have a great summer, and plan to have fun at the Regional Jamboree.<br />
</li><br />
<li><br />
Successfully complete and submit the iGEM 2013 Judging form.<br />
</li><br />
<li><br />
Create and share a Description of the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction">team's project</a> using the iGEM wiki and the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">team's parts using the Registry of Standard Biological Parts.</a><br />
</li><br />
<li><br />
Plan to present a Poster and Talk at the iGEM Jamboree.<br />
</li><br />
<li><br />
Document at least one new standard BioBrick Part or Device used in the project/central to your project and submit this part to the iGEM Registry.<br /><br />
We have constructed, characterized and submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">6 new Parts</a> helpful for the creation of a photo inducible fruit ripening control circuit, but not only. These are our entries:<br /><br />
<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, <a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>, <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. <BR><br />
<br />
We also have submitted many other useful parts, check our <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">complete list</a>.<br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="silver medal"><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Tn-2013Silver-medalaaaaa.png" /><br />
</div><br />
<div class="silver text"><br />
<span class="tn-subtitle">Additional Requirements for a Silver Medal:</span><br />
<ul><br />
<li><br />
Experimentally validate that at least one new BioBrick Part or Device of your own design and construction work as expected.<br /><br />
The device <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> was used to successfully produce ethylene and to ripen all of the fruit that we tested!<br><br />
The following parts built by us were also shown to work as expected: <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>.<br />
</li><br />
<li><br />
Document the characterization of this part in the 'Main Page' of that Part's/Device's Registry entry Submit this new part to the iGEM Parts Registry. <br />
</li> <br />
<li><br />
Describe in the wiki one or more ways in which the environment, security, safety and ethics and/or ownership and sharing or other broader implications have been taken into consideration in the design and execution of your project.<br /><br />
We have thoroughly analysed the possible pathways available in nature for ethylene production. The choice of the right pathway was done keeping always in mind safety and environmental issues. For example, we decided not to use the plant ethylene pathway, which would produce also cyanide...! Also <i>B. fruity</i> was designed with a photoinducible control system, to provide a safe alternative to commercial ripening methods that exploit ethylene. Check our <a href="https://2013.igem.org/Team:UNITN-Trento/Safety">Safety page</a> for specific info. <br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="gold medal"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png" /><br />
</div><br />
<div class="gold text"><br />
<span class="tn-subtitle">Additional Requirements for a Gold Medal: </span><br />
<ul><br />
<li><br />
Improve the function of an existing BioBrick Part or Device, enter this information in the 'Experience' section of that Parts's Registry entry, create a new registry page for the improved part, and submit this part to the iGEM Registry.<br /><br />
We have improved 1 existing BioBricks from the registry: <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>.<br /><br />
Our new entries for this part are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>); <br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>).<br />
</li><br />
</ul><br />
We also have resubmitted in pSB1C3 two parts from the <i>Eau de coli</i> MIT 2006 project that were discontinued. We hope that these parts will be included in the 2014 registry and will be soon available to many other teams.<br/><br />
Our entries for these parts are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065100">BBa_K1065100</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45119">BBa_J45119</a>);<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065103">BBa_K1065103</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45319">BBa_J45319</a>).<br />
</li><br />
</ul><br />
</li><br />
<li><br />
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br /><br />
We have designed an innovative way to provide the consumer with ready-to-eat fruit that exploits our engineered bacterial system. For more details visit the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">Vending Machine page</a>.<br />
</li><br />
</ul><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 />
</div><br />
</div><br />
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<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Home%20Edition"><br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Judging</span><br />
<br />
<div class="bronze medal"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Tn-2013Bronze-medalaaaaa.png" /><br />
</div><br />
<div class="bronze text"><br />
<span class="tn-subtitle">Requirements for a Bronze Medal:</span><br />
<ul class="apple-list"><br />
<li><br />
<a href="https://igem.org/Team.cgi">Register the team</a>, have a great summer, and plan to have fun at the Regional Jamboree.<br />
</li><br />
<li><br />
Successfully complete and submit the iGEM 2013 Judging form.<br />
</li><br />
<li><br />
Create and share a Description of the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction">team's project</a> using the iGEM wiki and the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">team's parts using the Registry of Standard Biological Parts.</a><br />
</li><br />
<li><br />
Plan to present a Poster and Talk at the iGEM Jamboree.<br />
</li><br />
<li><br />
Document at least one new standard BioBrick Part or Device used in the project/central to your project and submit this part to the iGEM Registry.<br /><br />
We have constructed, characterized and submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">6 new Parts</a> helpful for the creation of a photo inducible fruit ripening control circuit, but not only. These are our entries:<br /><br />
<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, <a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>, <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. <BR><br />
<br />
We also have submitted many other useful parts, check our <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">complete list</a>.<br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="silver medal"><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Tn-2013Silver-medalaaaaa.png" /><br />
</div><br />
<div class="silver text"><br />
<span class="tn-subtitle">Additional Requirements for a Silver Medal:</span><br />
<ul><br />
<li><br />
Experimentally validate that at least one new BioBrick Part or Device of your own design and construction work as expected.<br /><br />
The device <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> was used to produce ethylene and to ripen all of the fruit that we tested!<br><br />
The following parts built by us were also shown to work as expected: <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>.<br />
</li><br />
<li><br />
Document the characterization of this part in the 'Main Page' of that Part's/Device's Registry entry Submit this new part to the iGEM Parts Registry. <br />
</li> <br />
<li><br />
Describe in the wiki one or more ways in which the environment, security, safety and ethics and/or ownership and sharing or other broader implications have been taken into consideration in the design and execution of your project.<br /><br />
We have thoroughly analysed the possible pathways available in nature for ethylene production. The choice of the right pathway was done keeping always in mind safety and environmental issues. For example, we decided not to use the plant ethylene pathway, which would produce also cyanide...! Also <i>B. fruity</i> was designed with a photoinducible control system, to provide a safe alternative to commercial ripening methods that exploit ethylene. Check our <a href="https://2013.igem.org/Team:UNITN-Trento/Safety">Safety page</a> for specific info. <br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="gold medal"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png" /><br />
</div><br />
<div class="gold text"><br />
<span class="tn-subtitle">Additional Requirements for a Gold Medal: </span><br />
<ul><br />
<li><br />
Improve the function of an existing BioBrick Part or Device, enter this information in the 'Experience' section of that Parts's Registry entry, create a new registry page for the improved part, and submit this part to the iGEM Registry.<br /><br />
We have improved 1 existing BioBricks from the registry: <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>.<br /><br />
Our new entries for this part are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>); <br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>).<br />
</li><br />
</ul><br />
We also have resubmitted in pSB1C3 two parts from the <i>Eau de coli</i> MIT 2006 project that were discontinued. We hope that these parts will be included in the 2014 registry and will be soon available to many other teams.<br/><br />
Our entries for these parts are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065100">BBa_K1065100</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45119">BBa_J45119</a>);<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065103">BBa_K1065103</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45319">BBa_J45319</a>).<br />
</li><br />
</ul><br />
</li><br />
<li><br />
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br /><br />
We have designed an innovative way to provide the consumer with ready-to-eat fruit that exploits our engineered bacterial system. For more details visit the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">Vending Machine page</a>.<br />
</li><br />
</ul><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 />
</div><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Home%20Edition"><br />
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<!--PAGE--><br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Judging</span><br />
<br />
<div class="bronze medal"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Tn-2013Bronze-medalaaaaa.png" /><br />
</div><br />
<div class="bronze text"><br />
<span class="tn-subtitle">Requirements for a Bronze Medal:</span><br />
<ul class="apple-list"><br />
<li><br />
<a href="https://igem.org/Team.cgi">Register the team</a>, have a great summer, and plan to have fun at the Regional Jamboree.<br />
</li><br />
<li><br />
Successfully complete and submit the iGEM 2013 Judging form.<br />
</li><br />
<li><br />
Create and share a Description of the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction">team's project</a> using the iGEM wiki and the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">team's parts using the Registry of Standard Biological Parts.</a><br />
</li><br />
<li><br />
Plan to present a Poster and Talk at the iGEM Jamboree.<br />
</li><br />
<li><br />
Document at least one new standard BioBrick Part or Device used in the project/central to your project and submit this part to the iGEM Registry.<br /><br />
We have constructed, characterized and submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">6 new Parts</a> helpful for the creation of a photo inducible fruit ripening control circuit, but not only. These are our entries:<br /><br />
<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, <a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>, <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. <BR><br />
<br />
We also have submitted many other useful parts, check our <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">complete list</a>.<br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="silver medal"><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Tn-2013Silver-medalaaaaa.png" /><br />
</div><br />
<div class="silver text"><br />
<span class="tn-subtitle">Additional Requirements for a Silver Medal:</span><br />
<ul><br />
<li><br />
Experimentally validate that at least one new BioBrick Part or Device of your own design and construction work as expected.<br /><br />
The device <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> was used to produce ethylene and ripen all of the fruit that we tested!<br><br />
The following parts built by us were also shown to work as expected: <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>.<br />
</li><br />
<li><br />
Document the characterization of this part in the 'Main Page' of that Part's/Device's Registry entry Submit this new part to the iGEM Parts Registry. <br />
</li> <br />
<li><br />
Describe in the wiki one or more ways in which the environment, security, safety and ethics and/or ownership and sharing or other broader implications have been taken into consideration in the design and execution of your project.<br /><br />
We have thoroughly analysed the possible pathways available in nature for ethylene production. The choice of the right pathway was done keeping always in mind safety and environmental issues. For example, we decided not to use the plant ethylene pathway, which would produce also cyanide...! Also <i>B. fruity</i> was designed with a photoinducible control system, to provide a safe alternative to commercial ripening methods that exploit ethylene. Check our <a href="https://2013.igem.org/Team:UNITN-Trento/Safety">Safety page</a> for specific info. <br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="gold medal"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png" /><br />
</div><br />
<div class="gold text"><br />
<span class="tn-subtitle">Additional Requirements for a Gold Medal: </span><br />
<ul><br />
<li><br />
Improve the function of an existing BioBrick Part or Device, enter this information in the 'Experience' section of that Parts's Registry entry, create a new registry page for the improved part, and submit this part to the iGEM Registry.<br /><br />
We have improved 1 existing BioBricks from the registry: <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>.<br /><br />
Our new entries for this part are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>); <br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>).<br />
</li><br />
</ul><br />
We also have resubmitted in pSB1C3 two parts from the <i>Eau de coli</i> MIT 2006 project that were discontinued. We hope that these parts will be included in the 2014 registry and will be soon available to many other teams.<br/><br />
Our entries for these parts are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065100">BBa_K1065100</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45119">BBa_J45119</a>);<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065103">BBa_K1065103</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45319">BBa_J45319</a>).<br />
</li><br />
</ul><br />
</li><br />
<li><br />
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br /><br />
We have designed an innovative way to provide the consumer with ready-to-eat fruit that exploits our engineered bacterial system. For more details visit the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">Vending Machine page</a>.<br />
</li><br />
</ul><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 />
</div><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Home%20Edition"><br />
<img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/3/38/Tn-2013-arr-JUDGING_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/Team"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/7/72/Tn-2013-arr-JUDGING_next.png" /><br />
</a><br />
</div><br />
</div><br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/3/37/Tn-2013-headerbg-Sfondoor.jpg</html>|<html>https://static.igem.org/mediawiki/2013/8/8e/Tn-2013-headerbg-Sfondoor_or.jpg</html>}}</div>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/PartsTeam:UNITN-Trento/Parts2013-10-04T15:44:00Z<p>Cridelbianco: </p>
<hr />
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<link rel="stylesheet" href="https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/CSS/Parts&action=raw&ctype=text/css" type="text/css" /><br />
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<!-- Page --><br />
<div class="tn-parts-container"><br />
<div class="legend"><br />
<div class="sheet"><br />
<p><br />
In this beautiful page you can find <i>the fruits</i> of our summer’s efforts, otherwise you can just go to the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento" target="_blank">official page</a>.<br />
<p><br />
The number of bananas indicates:<br />
</p><br />
<ul><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: This is an intermediate part and it has not been characterized.</li><br />
<br />
<li><img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" />: the part does not work, due to a mistake of some kind, or we do not know if it works and more tests must be performed. </li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: the part was characterized but doesn't work always as expected.</li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: the part works as expected and has been characterized.</li><br />
<br />
</ul><br />
<p style="margin-bottom: 0;"><br />
<i>&#9835; &#9834; “One banana, two bananas, three bananas, four. Four bananas make a bunch and so do many more.” &#9834; &#9835;</i><br />
</p><br />
<p style="margin-top: 0; text-align: right;"><br />
[cit. The Banana Splits – <i>The Tra La La Song</i>]<br />
</p><br />
</div><br />
</div><br />
<div class="content"><br />
<!-- Ethylene --><br />
<div class="box"><br />
<div id="label1" class="label"><br />
</div><br />
<span class="title"><a id="eth">Ethylene</a></span><br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065000">BBa_K1065000</a></td><br />
<td class="name">EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Ethylene Forming Enzyme gene (EFE) that forms ethylene starting from 2-oxoglutarate.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065002">BBa_K1065002</a></td><br />
<td class="name">EFE with double-terminator</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE followed by the double terminator BBa_B0015.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065001">BBa_K1065001</a></td><br />
<td class="name">EFE arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is EFE followed by a double terminator (B0015) under the control of the araCpBAD inducible promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- Methyl salicylate --><br />
<div class="box"><br />
<div id="label2" class="label"><br />
</div><br />
<span class="title"><a id="mesa">Methyl salicylate</a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065100">BBa_K1065100</a></td><br />
<td class="name">BSMT1</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This part encodes BSMT1, an enzyme that methylates salycilic acid to obtain methyl salycilate. It is the original part from the '<i>Eau de coli</i>' MIT iGEM 2006 project, re-submitted (<i>and resurrected</i>) by us in pSB1C3.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065103">BBa_K1065103</a></td><br />
<td class="name">PchBA</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
PchA is the enzyme responsible for the formation of isochorismate from chorismate. The next step is performed by the PchB enzyme, which converts isochorismate in salicylic acid.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065104">BBa_K1065104</a></td><br />
<td class="name">SAM synthetase</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This part encodes the SAM (S-AdoMet) synthetase gene, which we friendly call "uncle SAM". SAM is a methyl donor required to produce MeSA and other methylated molecules.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065101">BBa_K1065101</a></td><br />
<td class="name">BSMT1 arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A methyltransferase (BSMT1) regulated by the araCpBAD promoter. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065105">BBa_K1065105</a></td><br />
<td class="name">SAM synthetase under pLac promoter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is SAM synthetase (<i>uncle SAM</i>) regulated by the constitutive Lac promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065102">BBa_K1065102</a></td><br />
<td class="name">PchBA under pLac and BSMT1 arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The wintergreen device: PchBA under a mutant Lac promoter (BBa_K1065107) followed by BSMT1 under the araCpBAD promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065106">BBa_K1065106</a></td><br />
<td class="name">BSMT1, PchBA and SAM synthetase</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
An improved version of the wintergreen device: BBa_K1065102 with SAM synthetase to boost production.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- Blue light --><br />
<div class="box"><br />
<div id="label3" class="label"><br />
</div><br />
<span class="title"><a id="blue">Blue light</a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065302">BBa_K1065302</a></td><br />
<td class="name">Blue light circuit to produce amilGFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A device to characterize our improved part BBa_K1065305 controlled by pLac promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065305">BBa_K1065305</a></td><br />
<td class="name">BBa_K952003 improved</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
No rbs, no party!!!!We improved BBa_K952003 by mutagenesis to add the missing RBS, now the part works. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065309">BBa_K1065309</a></td><br />
<td class="name">Blue light ethylene producing device</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Complete blue-light sensing circuit without inverter, producing both amilGFP and EFE.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065310">BBa_K1065310</a></td><br />
<td class="name">Complete blue-light sensing circuit with an inverter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Yf1 and FixJ are regulated by the Anderson promoter (BBa_J23100). pLambda regulates the expression of the reporter gene amilCP. When exposed to blue-light, amilCP is synthesized, when the blue-light is not present, amilCP is not produced.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065311">BBa_K1065311</a></td><br />
<td class="name">Complete blue-light sensing device with inverter, amilCP and EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Our complete circuit composed by:J23100, Yf1 and FixJ, pFixK2, CI, pLambda, amilCP and EFE.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065306">BBa_K1065306</a></td><br />
<td class="name">J23100+Yf1+FixJ</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Yf1 (photosensor) and FixJ (response regulator) under the Anderson promoter (BBa_J23100).<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065307">BBa_K1065307</a></td><br />
<td class="name">J23100+Yf1+FixJ (with terminators)</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The same as BBa_K1065306, just with the terminators at the end. This part was not submitted to the registry. If you are interested please contact us.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065308">BBa_K1065308</a></td><br />
<td class="name">BBa_K1065307 with inverter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is an intermediate part to build a blue light inducible genetic circuit with the inverter. This part is in pSB1A2 and it was not submitted to the registry. If you are interested in this part please contact us.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065303">BBa_K1065303</a></td><br />
<td class="name">Yf1+FixJ+pFixK2</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The starting point to build a blue-light sensing circuit: the photosensor (Yf1) and the response regulator (FixJ) with its promoter (pFixK2). This part was build merging BBa_K592016 and BBa_K592006.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065300">BBa_K1065300</a></td><br />
<td class="name">Blue light circuit to produce amilGFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
It does not work beacuse it lacks the RBS before the amilGFP gene.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- Bacillus --><br />
<div class="box"><br />
<div id="label4" class="label"><br />
</div><br />
<span class="title"><a id="bacillus"><i>Bacillus subtilis</i></a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065201">BBa_K1065201</a></td><br />
<td class="name">pXyl+GFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A <i>B. subtilis</i> xylose inducible plasmid (from LMU Munich iGEM 2012) to produce GFPmut3b, regulated by a strong ribosome binding site (Bba_e0840).<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065203">BBa_K1065203</a></td><br />
<td class="name">Efe+Bba_B0015 in pSpac.</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE in pSpac vector, specific for <i>B. subtilis</i>.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065204">BBa_K1065204</a></td><br />
<td class="name">pXyl+EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE in pXyl vector, specific for <i>B. subtilis</i>. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<div class="container"><br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Modeling"><br />
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<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/e/ef/Tn-2013-headerbg-Sfondogr2.jpg</html>|<html>https://static.igem.org/mediawiki/2013/5/52/Tn-2013-headerbg-Sfondogr2_OR.jpg</html>}}</div>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/PartsTeam:UNITN-Trento/Parts2013-10-04T15:43:19Z<p>Cridelbianco: </p>
<hr />
<div><!--NEW PAGE--><br />
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<br />
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<!-- Page --><br />
<div class="tn-parts-container"><br />
<div class="legend"><br />
<div class="sheet"><br />
<p><br />
In this beautiful page you can find <i>the fruits</i> of our summer’s efforts, otherwise you can just go to the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento" target="_blank">official page</a>.<br />
<p><br />
The number of bananas indicates:<br />
</p><br />
<ul><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: This is an intermediate part and it has not been characterized.</li><br />
<br />
<li><img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" />: the part does not work, due to a mistake of some kind, or we do not know if it works and more tests must be performed. </li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: the part was characterized but doesn't work always as espected.</li><br />
<li><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" />: the part works as expected and has been characterized.</li><br />
<br />
</ul><br />
<p style="margin-bottom: 0;"><br />
<i>&#9835; &#9834; “One banana, two bananas, three bananas, four. Four bananas make a bunch and so do many more.” &#9834; &#9835;</i><br />
</p><br />
<p style="margin-top: 0; text-align: right;"><br />
[cit. The Banana Splits – <i>The Tra La La Song</i>]<br />
</p><br />
</div><br />
</div><br />
<div class="content"><br />
<!-- Ethylene --><br />
<div class="box"><br />
<div id="label1" class="label"><br />
</div><br />
<span class="title"><a id="eth">Ethylene</a></span><br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065000">BBa_K1065000</a></td><br />
<td class="name">EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Ethylene Forming Enzyme gene (EFE) that forms ethylene starting from 2-oxoglutarate.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065002">BBa_K1065002</a></td><br />
<td class="name">EFE with double-terminator</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE followed by the double terminator BBa_B0015.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065001">BBa_K1065001</a></td><br />
<td class="name">EFE arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is EFE followed by a double terminator (B0015) under the control of the araCpBAD inducible promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- Methyl salicylate --><br />
<div class="box"><br />
<div id="label2" class="label"><br />
</div><br />
<span class="title"><a id="mesa">Methyl salicylate</a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065100">BBa_K1065100</a></td><br />
<td class="name">BSMT1</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This part encodes BSMT1, an enzyme that methylates salycilic acid to obtain methyl salycilate. It is the original part from the '<i>Eau de coli</i>' MIT iGEM 2006 project, re-submitted (<i>and resurrected</i>) by us in pSB1C3.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065103">BBa_K1065103</a></td><br />
<td class="name">PchBA</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
PchA is the enzyme responsible for the formation of isochorismate from chorismate. The next step is performed by the PchB enzyme, which converts isochorismate in salicylic acid.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065104">BBa_K1065104</a></td><br />
<td class="name">SAM synthetase</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This part encodes the SAM (S-AdoMet) synthetase gene, which we friendly call "uncle SAM". SAM is a methyl donor required to produce MeSA and other methylated molecules.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065101">BBa_K1065101</a></td><br />
<td class="name">BSMT1 arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A methyltransferase (BSMT1) regulated by the araCpBAD promoter. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065105">BBa_K1065105</a></td><br />
<td class="name">SAM synthetase under pLac promoter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is SAM synthetase (<i>uncle SAM</i>) regulated by the constitutive Lac promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065102">BBa_K1065102</a></td><br />
<td class="name">PchBA under pLac and BSMT1 arabinose-regulated</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The wintergreen device: PchBA under a mutant Lac promoter (BBa_K1065107) followed by BSMT1 under the araCpBAD promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065106">BBa_K1065106</a></td><br />
<td class="name">BSMT1, PchBA and SAM synthetase</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
An improved version of the wintergreen device: BBa_K1065102 with SAM synthetase to boost production.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- Blue light --><br />
<div class="box"><br />
<div id="label3" class="label"><br />
</div><br />
<span class="title"><a id="blue">Blue light</a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065302">BBa_K1065302</a></td><br />
<td class="name">Blue light circuit to produce amilGFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A device to characterize our improved part BBa_K1065305 controlled by pLac promoter.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065305">BBa_K1065305</a></td><br />
<td class="name">BBa_K952003 improved</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
No rbs, no party!!!!We improved BBa_K952003 by mutagenesis to add the missing RBS, now the part works. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065309">BBa_K1065309</a></td><br />
<td class="name">Blue light ethylene producing device</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Complete blue-light sensing circuit without inverter, producing both amilGFP and EFE.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065310">BBa_K1065310</a></td><br />
<td class="name">Complete blue-light sensing circuit with an inverter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Yf1 and FixJ are regulated by the Anderson promoter (BBa_J23100). pLambda regulates the expression of the reporter gene amilCP. When exposed to blue-light, amilCP is synthesized, when the blue-light is not present, amilCP is not produced.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065311">BBa_K1065311</a></td><br />
<td class="name">Complete blue-light sensing device with inverter, amilCP and EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Our complete circuit composed by:J23100, Yf1 and FixJ, pFixK2, CI, pLambda, amilCP and EFE.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065306">BBa_K1065306</a></td><br />
<td class="name">J23100+Yf1+FixJ</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
Yf1 (photosensor) and FixJ (response regulator) under the Anderson promoter (BBa_J23100).<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065307">BBa_K1065307</a></td><br />
<td class="name">J23100+Yf1+FixJ (with terminators)</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The same as BBa_K1065306, just with the terminators at the end. This part was not submitted to the registry. If you are interested please contact us.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065308">BBa_K1065308</a></td><br />
<td class="name">BBa_K1065307 with inverter</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
This is an intermediate part to build a blue light inducible genetic circuit with the inverter. This part is in pSB1A2 and it was not submitted to the registry. If you are interested in this part please contact us.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/10/Tn-2013-parts-Banana-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065303">BBa_K1065303</a></td><br />
<td class="name">Yf1+FixJ+pFixK2</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
The starting point to build a blue-light sensing circuit: the photosensor (Yf1) and the response regulator (FixJ) with its promoter (pFixK2). This part was build merging BBa_K592016 and BBa_K592006.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065300">BBa_K1065300</a></td><br />
<td class="name">Blue light circuit to produce amilGFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
It does not work beacuse it lacks the RBS before the amilGFP gene.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
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<span class="title"><a id="bacillus"><i>Bacillus subtilis</i></a></span><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065201">BBa_K1065201</a></td><br />
<td class="name">pXyl+GFP</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
A <i>B. subtilis</i> xylose inducible plasmid (from LMU Munich iGEM 2012) to produce GFPmut3b, regulated by a strong ribosome binding site (Bba_e0840).<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
<tr><br />
<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065203">BBa_K1065203</a></td><br />
<td class="name">Efe+Bba_B0015 in pSpac.</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE in pSpac vector, specific for <i>B. subtilis</i>.<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="part-box"><br />
<table><br />
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<td rowspan="2" class="grade"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1e/Tn-2013-parts-Banana-green-parts.png" /><br />
</td><br />
<td class="code"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1065204">BBa_K1065204</a></td><br />
<td class="name">pXyl+EFE</td><br />
</tr><br />
<tr><br />
<td colspan="2" class="descr"><br />
EFE in pXyl vector, specific for <i>B. subtilis</i>. <br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
</div><br />
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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 threated 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 threated 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 />
</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/Project/Bacillus"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/6/6e/Tn-2013-arr-Ripening_next.png" /><br />
</a><br />
</div><br />
</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 />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/8/8e/Tn-2013-headerbg-Sfondoor_or.jpg</html>|<html>https://static.igem.org/mediawiki/2013/3/37/Tn-2013-headerbg-Sfondoor.jpg</html>}}</div>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/Project/Blue_lightTeam:UNITN-Trento/Project/Blue light2013-10-04T14:31:31Z<p>Cridelbianco: </p>
<hr />
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<br />
<!--CSS--><br />
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<br />
<!-- PAGE --><br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Results - Blue Light</span><br />
<p><br />
We decided to develop a photoinducible genetic circuit that triggers the production of ethylene in the presence of blue light (470 nm), and blocks the production of ethylene in the dark.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/a/ab/Tn-2013_Photoreceptor_path.png" /><br />
<p><br />
We decided to use blue light as our inducer because a photoinducible system is highly compatible with our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine"><i>B. fruity </i> vending machine</a>. All parts have been transformed and characterized in <i>E. coli</i> NEB10&beta;.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013_Bluelight_viola_tom_br1.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/a/a7/Tn-2013_Bluelight_cate.jpg" /><br />
</div><br />
<br />
<span class="tn-subtitle">The device</span><br />
<p><br />
We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed a blue light dependent device that includes an inverter cassette.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013_bluelight_Constructs.jpg" /><br><br />
<p><br />
We engineered in <i>E. coli</i> a blue-light sensor composed of:<br />
</p><br />
<ul><br />
<li><br />
Anderson promoter BBa_J23100;<br />
</li><br />
<li><br />
the blue light receptor YF1, which consists of YtvA from <i>B. subtilis</i> fused to a kinase domain (FixL) from <i>B. japonicum</i>; <span class="tn-ref">(Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="tn-ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span><br />
</li><br />
<li><br />
its response regulator, FixJ;<br />
</li><br />
<li><br />
a downstream promoter pFixK2, which is turned off by phosphorylated FixJ;<br />
</li><br />
<li><br />
an inverter cassette composed of cI and pLambda;<br />
</li><br />
<li><br />
a reporter (chromoprotein amilCP), subsequently EFE was incorporated.<br />
</li><br />
</ul><br />
<br />
<p><br />
To assemble this device we used the following parts from the registry:<br />
</p><br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> from Berkeley 2006 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> from Uppsala 2011 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592020">BBa_K592020</a> from Uppsala 2011 iGEM team.<br />
</li><br />
</ul><br />
<br />
<p><br />
We characterized this circuit along with the version without the inverter cassette (activated in the dark and inhibited by blue light).<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br><br />
<br />
<span class="tn-subtitle">Different sources of blue light induces amilCP production in the "inverted circuit"</span><br />
<p><br />
We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.<br><br />
At first we compared the induction power of several light sources (<b>Figure 1</b>):<br />
</p><br />
<ul><br />
<li><br />
1 LED blue light;<br />
</li><br />
<li><br />
1 blue light bulb;<br />
</li><br />
<li><br />
1 white light bulb.<br />
</li><br />
</ul><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/7/73/Tn-2013_Diff_lights_induction.jpg" /><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/e/e8/Tn-2013_different_lights_pic.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 1: Different light sources induction power.</b> We had massive production of amilCP with LED light (4) and white light (3). Instead exposure to the blue light bulb (2) induced a little less but still much more than the dark control (1): probably white light worked as well because white light contains the right wavelength (470 nm) while the blue bulb wavelength range is unknown.</span><br />
<br />
<p><br />
Therefore we decided to use only blue LED and normal light as inducers in further tests (<b>Figure 2</b>).<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_bluelight_inverte_pic2.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/ba/Tn-2013_bluelight_noinverter_pic2.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> We started the characterization test by splitting the cultures at an O.D. of 0.7 into 3 put at 3 different conditions: blue LED induced culture (1), normal light induced culture (2), dark control (3). We induced O/N at 37&deg;C. <b>After overnight incubation the differences in the cultures were very clear</b>.</span><br />
<br />
<p><br />
Moreover, amilCP has an absorbance peak at 588 nm so we measured the absorbance peak at the UV-VIS spectrometer (PerkinElmer lambda 25) in order to have more quantitative data (<b>Figure 3</b>). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/Absorbance_blue_light_fabio.png" /><br />
<span class="tn-caption"><b>Fig. 3:</b> by measuring the absorbance of the three samples (588 nm), we observed a substantial difference between the dark control and the other two samples. This is a quantitative demonstration that the production of amilCP in the dark was strongly repressed. The graph also shows that white light induction is a little less efficient.</span><br />
<br />
<p><br />
We carried out several tests in order to demonstrate the reproducibility of the behavior. Sometimes we observed amilCP production even in the dark control. Therefore it seems that the circuit doesn’t act like a perfectly controlled switch and/or there are other unidentified variables that impact the system. We can speculate on some potential causes: <br />
<ul><br />
<li>the inverter cassette may not function as expected;</li><br />
<li>not enough cI is produced due to its position in the circuitry.</li><br />
</ul><br />
To further probe our system <b>we tested also the circuit without the inverter</b>. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a> which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_Norbs_yellowpart.jpeg" /><br />
<p><br />
The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We <b>improved this part</b> by inserting the missing RBS via mutagenic PCR.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/2/20/Tn-2013_Rbs_yellowpart.jpeg" /><br />
<p><br />
In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br />
<br />
<span class="tn-subtitle">Better defined switch observed in the circuit without inverter</span><br />
<p><br />
In order to demonstrate the actual enhancement of the device we induced both the improved circuit and the original part (to test it we created the part <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>). So we compared samples depending on two factors: induction/non induction and RBS/no-RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that is excitated at 503 nm and emits at 512 nm. For measurements we resuspended sonicated sample pellets in 2 ml of PBS.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg" /><br />
</div><br />
<span class="tn-caption"><b> Fig. 4: Yellow shade appears only in the induced sample with RBS: data confirmed by fluorescence measurements</b>. In the left panel, fluorescence spectra show that <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> induced sample (purple) produces amilGFP and the control sample (green) shows a basal expression. As expected the part without the RBS <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a> doesn't produce amilGFP neither in the dark (blue) nor under illumination (red). Right panel: after the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 10ml at 37°C: induced sample in the dark (1) and blue light exposed control (2). We also made 2 samples (3 and 4) in the same conditions from a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>, in order to compare the original part to the improved one. amilGFP is produced only in the induced sample with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>.<b> Our part with RBS is undeniably improved and works as expected</b>.</span><br />
<br />
<span class="tn-title">Summary</span><br />
<p><br />
<b>We achieved a successful characterization of both circuits with and without the inverter</b>. There was a substantial difference between the controls and the induced samples. There were some issues in implementing <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. Nevertheless, the construct that did not contain the inverter (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>) provided the desired functionality.<br />
<p><br />
<p><br />
There are a few things that we can try to improve the device such as adding a terminator after pFixK2, substituting J23100 with pLac promoter or improving the transcription of cI. We are working on this now.<br />
</p><br />
<br />
<span class="tn-effect"><br />
We used this circuit to build a device that produces ethylene in response to blue light. For more info check our ethylene page<br />
</span><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Methyl%20Salicylate"><br />
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<a href="javascript:toTop('#tn-main-wrap-wrap');"><br />
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</a><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/4/49/Tn-2013-arr-BlueLight_next.png" /><br />
</a><br />
</div><br />
</div><br />
<br />
<a id="tn-sp-tour" href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening#tour"><br />
<img src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-tour-G_AAA_DSC_0061.png" /><br />
<span>Continue the tour!</span><br />
</a><br />
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<!--end content--><br />
</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>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/Project/Blue_lightTeam:UNITN-Trento/Project/Blue light2013-10-04T14:30:41Z<p>Cridelbianco: </p>
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<!-- PAGE --><br />
<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Results - Blue Light</span><br />
<p><br />
We decided to develop a photoinducible genetic circuit that triggers the production of ethylene in the presence of blue light (470 nm), and blocks the production of ethylene in the dark.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/a/ab/Tn-2013_Photoreceptor_path.png" /><br />
<p><br />
We decided to use blue light as our inducer because a photoinducible system is highly compatible with our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine"><i>B. fruity </i> vending machine</a>. All parts have been transformed and characterized in <i>E. coli</i> NEB10&beta;.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013_Bluelight_viola_tom_br1.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/a/a7/Tn-2013_Bluelight_cate.jpg" /><br />
</div><br />
<br />
<span class="tn-subtitle">The device</span><br />
<p><br />
We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed a blue light dependent device that includes an inverter cassette.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013_bluelight_Constructs.jpg" /><br><br />
<p><br />
We engineered in <i>E. coli</i> a blue-light sensor composed of:<br />
</p><br />
<ul><br />
<li><br />
Anderson promoter BBa_J23100;<br />
</li><br />
<li><br />
the blue light receptor YF1, which consists of YtvA from <i>B. subtilis</i> fused to a kinase domain (FixL) from <i>B. japonicum</i>; <span class="tn-ref">(Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="tn-ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span><br />
</li><br />
<li><br />
its response regulator, FixJ;<br />
</li><br />
<li><br />
a downstream promoter pFixK2, which is turned off by phosphorylated FixJ;<br />
</li><br />
<li><br />
an inverter cassette composed of cI and pLambda;<br />
</li><br />
<li><br />
a reporter (chromoprotein amilCP), subsequently EFE was incorporated.<br />
</li><br />
</ul><br />
<br />
<p><br />
To assemble this device we used the following parts from the registry:<br />
</p><br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> from Berkeley 2006 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> from Uppsala 2011 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592020">BBa_K592020</a> from Uppsala 2011 iGEM team.<br />
</li><br />
</ul><br />
<br />
<p><br />
We characterized this circuit along with the version without the inverter cassette (activated in the dark and inhibited by blue light).<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br><br />
<br />
<span class="tn-subtitle">Different sources of blue light induces amilCP production in the "inverted circuit"</span><br />
<p><br />
We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.<br><br />
At first we compared the induction power of several light sources (<b>Figure 1</b>):<br />
</p><br />
<ul><br />
<li><br />
1 LED blue light;<br />
</li><br />
<li><br />
1 blue light bulb;<br />
</li><br />
<li><br />
1 white light bulb.<br />
</li><br />
</ul><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/7/73/Tn-2013_Diff_lights_induction.jpg" /><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/e/e8/Tn-2013_different_lights_pic.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 1: Different light sources induction power.</b> We had massive production of amilCP with LED light (4) and white light (3). Instead exposure to the blue light bulb (2) induced a little less but still much more than the dark control (1): probably white light worked as well because white light contains the right wavelength (470 nm) while the blue bulb wavelength range is unknown.</span><br />
<br />
<p><br />
Therefore we decided to use only blue LED and normal light as inducers in further tests (<b>Figure 2</b>).<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_bluelight_inverte_pic2.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/ba/Tn-2013_bluelight_noinverter_pic2.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> We started the characterization test by splitting the cultures at an O.D. of 0.7 into 3 put at 3 different conditions: blue LED induced culture (1), normal light induced culture (2), dark control (3). We induced O/N at 37&deg;C. <b>After overnight incubation the differences in the cultures were very clear</b>.</span><br />
<br />
<p><br />
Moreover, amilCP has an absorbance peak at 588 nm so we measured the absorbance peak at the UV-VIS spectrometer (PerkinElmer lambda 25) in order to have more quantitative data (<b>Figure 3</b>). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/Absorbance_blue_light_fabio.png" /><br />
<span class="tn-caption"><b>Fig. 3:</b> by measuring the absorbance of the three samples (588 nm), we observed a substantial difference between the dark control and the other two samples. This is a quantitative demonstration that the production of amilCP in the dark was strongly repressed. The graph also shows that white light induction is a little less efficient.</span><br />
<br />
<p><br />
We carried out several tests in order to demonstrate the reproducibility of the behavior. Sometimes we observed amilCP production even in the dark control. Therefore it seems that the circuit doesn’t act like a perfectly controlled switch and/or there are other unidentified variables that impact the system. We can speculate on some potential causes: <br />
<ul><br />
<li>the inverter cassette may not function as expected;</li><br />
<li>not enough cI is produced due to its position in the circuitry.</li><br />
</ul><br />
To further probe our system <b>we tested also the circuit without the inverter</b>. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a> which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_Norbs_yellowpart.jpeg" /><br />
<p><br />
The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We <b>improved this part</b> by inserting the missing RBS via mutagenic PCR.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/2/20/Tn-2013_Rbs_yellowpart.jpeg" /><br />
<p><br />
In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br />
<br />
<span class="tn-subtitle">Better defined switch observed in the circuit without inverter</span><br />
<p><br />
In order to demonstrate the actual enhancement of the device we induced both the improved circuit and the original part (to test it we created the part <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>). So we compared samples depending on two factors: induction/non induction and RBS/no-RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that is excitated at 503 nm and emits at 512 nm. For measurements we resuspended sonicated sample pellets in 2 ml of PBS.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg" /><br />
</div><br />
<span class="tn-caption"><b> Fig. 4: Yellow shade appears only in the induced sample with RBS: data confirmed by fluorescence measurements</b>. In the left panel, fluorescence spectra show that <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> induced sample (purple) produces amilGFP and the control sample (green) shows a basal expression. As expected the part without the RBS <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a> doesn't produce amilGFP neither in the dark (blue) nor under illumination (red). Right panel: after the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 10ml at 37°C: induced sample in the dark (1) and blue light exposed control (2). We also made 2 samples (3 and 4) in the same conditions from a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>, in order to compare the original part to the improved one. amilGFP is produced only in the induced sample with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>.<b>Our part with RBS is undeniably improved and works as expected</b>.</span><br />
<br />
<span class="tn-title">Summary</span><br />
<p><br />
<b>We achieved a successful characterization of both circuits with and without the inverter</b>. There was a substantial difference between the controls and the induced samples. There were some issues in implementing <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. Nevertheless, the construct that did not contain the inverter (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>) provided the desired functionality.<br />
<p><br />
<p><br />
There are a few things that we can try to improve the device such as adding a terminator after pFixK2, substituting J23100 with pLac promoter or improving the transcription of cI. We are working on this now.<br />
</p><br />
<br />
<span class="tn-effect"><br />
We used this circuit to build a device that produces ethylene in response to blue light. For more info check our ethylene page<br />
</span><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Methyl%20Salicylate"><br />
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</a><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f9/Tn-2013-tour-G_AAA_DSC_0061.png" /><br />
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<div class="sheet"><br />
<span class="tn-title">Results - Blue Light</span><br />
<p><br />
We decided to develop a photoinducible genetic circuit that triggers the production of ethylene in the presence of blue light (470 nm), and blocks the production of ethylene in the dark.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/a/ab/Tn-2013_Photoreceptor_path.png" /><br />
<p><br />
We decided to use blue light as our inducer because a photoinducible system is highly compatible with our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine"><i>B. fruity </i> vending machine</a>. All parts have been transformed and characterized in <i>E. coli</i> NEB10&beta;.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013_Bluelight_viola_tom_br1.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/a/a7/Tn-2013_Bluelight_cate.jpg" /><br />
</div><br />
<br />
<span class="tn-subtitle">The device</span><br />
<p><br />
We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed a blue light dependent device that includes an inverter cassette.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013_bluelight_Constructs.jpg" /><br><br />
<p><br />
We engineered in <i>E. coli</i> a blue-light sensor composed of:<br />
</p><br />
<ul><br />
<li><br />
Anderson promoter BBa_J23100;<br />
</li><br />
<li><br />
the blue light receptor YF1, which consists of YtvA from <i>B. subtilis</i> fused to a kinase domain (FixL) from <i>B. japonicum</i>; <span class="tn-ref">(Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="tn-ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span><br />
</li><br />
<li><br />
its response regulator, FixJ;<br />
</li><br />
<li><br />
a downstream promoter pFixK2, which is turned off by phosphorylated FixJ;<br />
</li><br />
<li><br />
an inverter cassette composed of cI and pLambda;<br />
</li><br />
<li><br />
a reporter (chromoprotein amilCP), subsequently EFE was incorporated.<br />
</li><br />
</ul><br />
<br />
<p><br />
To assemble this device we used the following parts from the registry:<br />
</p><br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> from Berkeley 2006 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> from Uppsala 2011 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592020">BBa_K592020</a> from Uppsala 2011 iGEM team.<br />
</li><br />
</ul><br />
<br />
<p><br />
We characterized this circuit along with the version without the inverter cassette (activated in the dark and inhibited by blue light).<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br><br />
<br />
<span class="tn-subtitle">Different sources of blue light induces amilCP production in the "inverted circuit"</span><br />
<p><br />
We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.<br><br />
At first we compared the induction power of several light sources (<b>Figure 1</b>):<br />
</p><br />
<ul><br />
<li><br />
1 LED blue light;<br />
</li><br />
<li><br />
1 blue light bulb;<br />
</li><br />
<li><br />
1 white light bulb.<br />
</li><br />
</ul><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/7/73/Tn-2013_Diff_lights_induction.jpg" /><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/e/e8/Tn-2013_different_lights_pic.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 1: Different light sources induction power.</b> We had massive production of amilCP with LED light (4) and white light (3). Instead exposure to the blue light bulb (2) induced a little less but still much more than the dark control (1): probably white light worked as well because white light contains the right wavelength (470 nm) while the blue bulb wavelength range is unknown.</span><br />
<br />
<p><br />
Therefore we decided to use only blue LED and normal light as inducers in further tests (<b>Figure 2</b>).<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_bluelight_inverte_pic2.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/ba/Tn-2013_bluelight_noinverter_pic2.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> We started the characterization test by splitting the cultures at an O.D. of 0.7 into 3 put at 3 different conditions: blue LED induced culture (1), normal light induced culture (2), dark control (3). We induced O/N at 37&deg;C. <b>After overnight incubation the differences in the cultures were very clear</b>.</span><br />
<br />
<p><br />
Moreover, amilCP has an absorbance peak at 588 nm so we measured the absorbance peak at the UV-VIS spectrometer (PerkinElmer lambda 25) in order to have more quantitative data (<b>Figure 3</b>). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/Absorbance_blue_light_fabio.png" /><br />
<span class="tn-caption"><b>Fig. 3:</b> by measuring the absorbance of the three samples (588 nm), we observed a substantial difference between the dark control and the other two samples. This is a quantitative demonstration that the production of amilCP in the dark was strongly repressed. The graph also shows that white light induction is a little less efficient.</span><br />
<br />
<p><br />
We carried out several tests in order to demonstrate the reproducibility of the behavior. Sometimes we observed amilCP production even in the dark control. Therefore it seems that the circuit doesn’t act like a perfectly controlled switch and/or there are other unidentified variables that impact the system. We can speculate on some potential causes: <br />
<ul><br />
<li>the inverter cassette may not function as expected;</li><br />
<li>not enough cI is produced due to its position in the circuitry.</li><br />
</ul><br />
To further probe our system <b>we tested also the circuit without the inverter</b>. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a> which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_Norbs_yellowpart.jpeg" /><br />
<p><br />
The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We <b>improved this part</b> by inserting the missing RBS via mutagenic PCR.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/2/20/Tn-2013_Rbs_yellowpart.jpeg" /><br />
<p><br />
In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br />
<br />
<span class="tn-subtitle">Better defined switch observed in the circuit without inverter</span><br />
<p><br />
In order to demonstrate the actual enhancement of the device we induced both the improved circuit and the original part (to test it we created the part <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>). So we compared samples depending on two factors: induction/non induction and RBS/no-RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that is excitated at 503 nm and emits at 512 nm. For measurements we resuspended sonicated sample pellets in 2 ml of PBS.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4: Yellow shade appears only in the induced sample with RBS: data confirmed by fluorescence measurements</b>.In the left panel, fluorescence spectra show that <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> induced sample (purple) produces amilGFP and the control sample (green) shows a basal expression. As expected the part without the RBS <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a> doesn't produce amilGFP neither in the dark (blue) nor under illumination (red). Right panel: after the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 10ml at 37°C: induced sample in the dark (1) and blue light exposed control (2). We also made 2 samples (3 and 4) in the same conditions from a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>, in order to compare the original part to the improved one. amilGFP is produced only in the induced sample with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>.<br><b>Our part with RBS is undeniably improved and works as expected</b>.</span><br />
<br />
<span class="tn-title">Summary</span><br />
<p><br />
<b>We achieved a successful characterization of both circuits with and without the inverter</b>. There was a substantial difference between the controls and the induced samples. There were some issues in implementing <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. Nevertheless, the construct that did not contain the inverter (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>) provided the desired functionality.<br />
<p><br />
<p><br />
There are a few things that we can try to improve the device such as adding a terminator after pFixK2, substituting J23100 with pLac promoter or improving the transcription of cI. We are working on this now.<br />
</p><br />
<br />
<span class="tn-effect"><br />
We used this circuit to build a device that produces ethylene in response to blue light. For more info check our ethylene page<br />
</span><br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Methyl%20Salicylate"><br />
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<span class="tn-title">Results - Blue Light</span><br />
<p><br />
We decided to develop a photoinducible genetic circuit that triggers the production of ethylene in the presence of blue light (470 nm), and blocks the production of ethylene in the dark.<br />
</p><br />
<br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/a/ab/Tn-2013_Photoreceptor_path.png" /><br />
<p><br />
We decided to use blue light as our inducer because a photoinducible system is highly compatible with our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine"><i>B. fruity </i> vending machine</a>. All parts have been transformed and characterized in <i>E. coli</i> NEB10&beta;.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/f/fb/Tn-2013_Bluelight_viola_tom_br1.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/a/a7/Tn-2013_Bluelight_cate.jpg" /><br />
</div><br />
<br />
<span class="tn-subtitle">The device</span><br />
<p><br />
We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed a blue light dependent device that includes an inverter cassette.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013_bluelight_Constructs.jpg" /><br><br />
<p><br />
We engineered in <i>E. coli</i> a blue-light sensor composed of:<br />
</p><br />
<ul><br />
<li><br />
Anderson promoter BBa_J23100;<br />
</li><br />
<li><br />
the blue light receptor YF1, which consists of YtvA from <i>B. subtilis</i> fused to a kinase domain (FixL) from <i>B. japonicum</i>; <span class="tn-ref">(Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="tn-ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span><br />
</li><br />
<li><br />
its response regulator, FixJ;<br />
</li><br />
<li><br />
a downstream promoter pFixK2, which is turned off by phosphorylated FixJ;<br />
</li><br />
<li><br />
an inverter cassette composed of cI and pLambda;<br />
</li><br />
<li><br />
a reporter (chromoprotein amilCP), subsequently EFE was incorporated.<br />
</li><br />
</ul><br />
<br />
<p><br />
To assemble this device we used the following parts from the registry:<br />
</p><br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> from Berkeley 2006 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> from Uppsala 2011 iGEM team;<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K592020">BBa_K592020</a> from Uppsala 2011 iGEM team.<br />
</li><br />
</ul><br />
<br />
<p><br />
We characterized this circuit along with the version without the inverter cassette (activated in the dark and inhibited by blue light).<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br><br />
<br />
<span class="tn-subtitle">Different sources of blue light induces amilCP production in the "inverted circuit"</span><br />
<p><br />
We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.<br><br />
At first we compared the induction power of several light sources (<b>Figure 1</b>):<br />
</p><br />
<ul><br />
<li><br />
1 LED blue light;<br />
</li><br />
<li><br />
1 blue light bulb;<br />
</li><br />
<li><br />
1 white light bulb.<br />
</li><br />
</ul><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/7/73/Tn-2013_Diff_lights_induction.jpg" /><br />
<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/e/e8/Tn-2013_different_lights_pic.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 1: Different light sources induction power.</b> We had massive production of amilCP with LED light (4) and white light (3). Instead exposure to the blue light bulb (2) induced a little less but still much more than the dark control (1): probably white light worked as well because white light contains the right wavelength (470 nm) while the blue bulb wavelength range is unknown.</span><br />
<br />
<p><br />
Therefore we decided to use only blue LED and normal light as inducers in further tests (<b>Figure 2</b>).<br />
</p><br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_bluelight_inverte_pic2.jpg" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/ba/Tn-2013_bluelight_noinverter_pic2.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> We started the characterization test by splitting the cultures at an O.D. of 0.7 into 3 put at 3 different conditions: blue LED induced culture (1), normal light induced culture (2), dark control (3). We induced O/N at 37&deg;C. <b>After overnight incubation the differences in the cultures were very clear</b>.</span><br />
<br />
<p><br />
Moreover, amilCP has an absorbance peak at 588 nm so we measured the absorbance peak at the UV-VIS spectrometer (PerkinElmer lambda 25) in order to have more quantitative data (<b>Figure 3</b>). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/Absorbance_blue_light_fabio.png" /><br />
<span class="tn-caption"><b>Fig. 3:</b> by measuring the absorbance of the three samples (588 nm), we observed a substantial difference between the dark control and the other two samples. This is a quantitative demonstration that the production of amilCP in the dark was strongly repressed. The graph also shows that white light induction is a little less efficient.</span><br />
<br />
<p><br />
We carried out several tests in order to demonstrate the reproducibility of the behavior. Sometimes we observed amilCP production even in the dark control. Therefore it seems that the circuit doesn’t act like a perfectly controlled switch and/or there are other unidentified variables that impact the system. We can speculate on some potential causes: <br />
<ul><br />
<li>the inverter cassette may not function as expected;</li><br />
<li>not enough cI is produced due to its position in the circuitry.</li><br />
</ul><br />
To further probe our system <b>we tested also the circuit without the inverter</b>. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a> which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K952003">BBa_K952003</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_Norbs_yellowpart.jpeg" /><br />
<p><br />
The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We <b>improved this part</b> by inserting the missing RBS via mutagenic PCR.<br />
</p><br />
<br />
<span class="tn-sub-subtitle"><a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a></span><br />
<img src="https://static.igem.org/mediawiki/2013/2/20/Tn-2013_Rbs_yellowpart.jpeg" /><br />
<p><br />
In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br />
<br />
<span class="tn-subtitle">Better defined switch observed in the circuit without inverter</span><br />
<p><br />
In order to demonstrate the actual enhancement of the device we induced both the improved circuit and the original part (to test it we created the part <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>). So we compared samples depending on two factors: induction/non induction and RBS/no-RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that is excitated at 503 nm and emits at 512 nm. For measurements we resuspended sonicated sample pellets in 2 ml of PBS.<br />
</p><br />
<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png" /><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg" /><br />
</div><br />
<span class="tn-caption"><b>Fig. 4: Yellow shade appears only in the induced sample with RBS: data confirmed by fluorescence measurements</b>.<br>In the left panel, fluorescence spectra show that <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> induced sample (purple) produces amilGFP and the control sample (green) shows a basal expression. As expected the part without the RBS doesn't produce amilGFP neither in the dark (blue) nor under illumination (red). Right panel: after the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 10ml at 37°C: induced sample in the dark (1) and blue light exposed control(2). We also made 2 sample (3 and 4) at the same conditions from a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>, in order to compare the original part to the improved one. amilGFP is produced only in the induced sample with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>.<br><b>Our part with RBS is undeniably improved and works as expected</b>.</span><br />
<br />
<span class="tn-title">Summary</span><br />
<p><br />
<b>We achieved a successful characterization of both circuits with and without the inverter</b>. There was a substantial difference between the controls and the induced samples. There were some issues in implementing <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. Nevertheless, the construct that did not contain the inverter (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>) provided the desired functionality.<br />
<p><br />
<p><br />
There are a few things that we can try to improve the device such as adding a terminator after pFixK2, substituting J23100 with pLac promoter or improving the transcription of cI. We are working on this now.<br />
</p><br />
<br />
<span class="tn-effect"><br />
We used this circuit to build a device that produces ethylene in response to blue light. For more info check our ethylene page<br />
</span><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) 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 />
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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 (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 />
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 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 (data not shown).<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>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <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 />
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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 blue light page 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. We did not have the possibility yet to test it for ethylene production. However 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-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 />
Jar volume (ml)<br />
</th><br />
<th><br />
Air volume in the jar + connections (ml)<br />
</th><br />
<th><br />
Culture volume (ml)<br />
</th><br />
<th><br />
Air/culture volumes ratio<br />
</th><br />
<th><br />
Concentration Expected<br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
500 <br />
</td><br />
<td><br />
800<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
2.66<br />
</td><br />
<td><br />
150.37<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
1000<br />
</td> <br />
<td><br />
1300<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
4.33<br />
</td><br />
<td><br />
92.37<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
1500<br />
</td> <br />
<td><br />
1800<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
6<br />
</td><br />
<td><br />
66.66<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 6:</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 blue light page 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. We did not have the possibility yet to test it for ethylene production. However 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 detected. <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-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 />
Jar volume (ml)<br />
</th><br />
<th><br />
Air volume in the jar + connections (ml)<br />
</th><br />
<th><br />
Culture volume (ml)<br />
</th><br />
<th><br />
Air/culture volumes ratio<br />
</th><br />
<th><br />
Concentration Expected<br />
</th> <br />
</tr> <br />
<tr><br />
<td><br />
500 <br />
</td><br />
<td><br />
800<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
2.66<br />
</td><br />
<td><br />
150.37<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
1000<br />
</td> <br />
<td><br />
1300<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
4.33<br />
</td><br />
<td><br />
92.37<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
1500<br />
</td> <br />
<td><br />
1800<br />
</td><br />
<td><br />
300<br />
</td><br />
<td><br />
6<br />
</td><br />
<td><br />
66.66<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 6:</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">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 maturation. Ethylene is an horomone 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 succesfully 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 />
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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 maturation. Ethylene is an horomone 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 succesfully 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 />
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Bacillus subtilis<br />
</span><br />
When we first came up with the idea of <i>B. fruity</i>, we immediatly thought that <i>B .subtilis</i> was the perfect chassis for a possible marketable application:<br />
<ol><br />
<li><br />
<i>Bacillus subtilis</i> sporulates and it can be stored in a inactive state;<br />
</li> <br />
<li><br />
<i>Bacillus subtilis</i> is not pathogenic and therefore can be used safely for food applications; <br />
</li><br />
<br />
</ol><br />
<br />
<i>Bacillus subtilis</i> would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a <i>B. fruity</i> home edition that exploits this principle. <br/><br />
To achieve this goal we started working with EFE, a ethylene forming enzyme from <i>Pseudomas Syringae</i> pv. phaseolicola (<a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>), which were inserted into pSBBs0K-Pspac (IPGT inducible) and pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for <i>B. subtilis</i> by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were kindly sent to us from LMU Munich).<br />
<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/><br />
<br />
<span class="tn-subtitle">Cloning of BBa_K1065203</span><br />
The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into <i>B. subtilis</i> genome was confirmed with the threonine assay.<br />
<div class="tn-doublephoto-wrap"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b0/Tn-2013_PXyl_digestion.png"/><br />
<img class="photo" src="https://static.igem.org/mediawiki/2013/3/3b/Tn-2013_thr_assay.jpg"/><br />
</div><br />
<span class="tn-caption"><b>Figure 1:</b> transformation of <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_1065204</a> in <i>B. subtilis</i>. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into <i>B. subtilis</i> 168 using minimal medium. Correct integration was confirmed with the threonine test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine.</span><br />
<br />
<span class="tn-subtitle">Toxicity assay</span><br />
We then measured the optical density of cells induced and non induced for both constructs.<br />
<div class="tn-doublephoto-wrap"><br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/8/8e/Tn-2013_K1065203_plot.png"> <br />
<img class="plot" src="https://static.igem.org/mediawiki/2013/1/14/Tn-2013_K1065204_plot.png"> <br />
</div><br />
<span class="tn-caption"><b>Figure 2:</b> <i>B. subtilis</i> 168 cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> or <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> were grown until an OD=0.9 and then splitted in two samples before induction. Cells were induced with 1% xylose for BBa_K1065203 and 0.5 mM of IPTG for BBa_K1065204. In both cases the induced samples (blue trace) grow slightly slower than the controls (red trace).</span><br />
<span class="tn-subtitle">Sporulation assay</span><br />
Spores were obtained by growing the transformed <i>B. subtilis</i> 168 cells in DSM medium, subjecting them to a heat shock at 60 &deg;C and plating them on a preheated glass slide. Spores were visualized at the microscope.<br />
<span class="tn-subtitle">Ethylene detection</span><br />
Ethylene production was tested by Gas Chromatography as we previoulsy did for <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>. The experiment was performed both from cultures started from fresh plates and from dry spores.<br/><br />
We did not observe any production of ethylene after 4 hours, nor after overnight induction. <br/><br />
At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip. <br />
<img src="https://static.igem.org/mediawiki/2013/6/69/Tn-2013_Lead-acetate_strip_assay.jpg"/><br />
<span style="text-align:justify;"class="tn-caption center"><b>Figure 4:</b> detection of sulfur compounds. <i>B. subtilis</i> 168 cells non transformed (1 and 2), transformed with <a href="http://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> and transformed with <a href="http://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1% xylose or with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate. </span><br />
<span class="tn-subtitle">Future directions</span><br />
For future experiments and improvement of the system we have identified additional potential drawbacks, including:<br />
<ul><br />
<li>pXyl could be inhibited by glucose although the threonine test confirmed the correct insertion of the vector;</li><br />
<li>the acquisition of pSpac could not be confirmed by colony PCR yet; even if the growth of colonies in the presence of the antibiotic indicates that the episomal vector carrying EFE gene is present;</li><br />
<li>at present the expression of the EFE gene has not been demonstrated. We plan to perform an additional real-time PCR experiment (to assess transcription).</li><br />
</ul> <br />
</div><br />
<br />
<br />
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<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit%20ripening"><br />
<img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/4/46/Tn-2013-arr-Bacillus_prev.png" /><br />
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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 />
<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 />
</div><br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene"><br />
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<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/2/26/Tn-2013-arr-MeSA_next.png" /><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>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/Extra/JudgingTeam:UNITN-Trento/Extra/Judging2013-10-04T08:30:45Z<p>Cridelbianco: </p>
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<span class="tn-title">Judging</span><br />
<br />
<div class="bronze medal"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e9/Tn-2013Bronze-medalaaaaa.png" /><br />
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<div class="bronze text"><br />
<span class="tn-subtitle">Requirements for a Bronze Medal:</span><br />
<ul class="apple-list"><br />
<li><br />
<a href="https://igem.org/Team.cgi">Register the team</a>, have a great summer, and plan to have fun at the Regional Jamboree.<br />
</li><br />
<li><br />
Successfully complete and submit the iGEM 2013 Judging form.<br />
</li><br />
<li><br />
Create and share a Description of the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Introduction">team's project</a> using the iGEM wiki and the <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">team's parts using the Registry of Standard Biological Parts.</a><br />
</li><br />
<li><br />
Plan to present a Poster and Talk at the iGEM Jamboree.<br />
</li><br />
<li><br />
Document at least one new standard BioBrick Part or Device used in the project/central to your project and submit this part to the iGEM Registry.<br /><br />
We have constructed, characterized and submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">6 new Parts</a> helpful for the creation of a photo inducible fruit ripening control circuit, but not only. These are our entries:<br /><br />
<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, <a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>, <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a>, <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. <BR><br />
<br />
We also have submitted many other useful parts, check our <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UNITN-Trento">complete list</a>.<br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="silver medal"><br />
<img src="https://static.igem.org/mediawiki/2013/3/31/Tn-2013Silver-medalaaaaa.png" /><br />
</div><br />
<div class="silver text"><br />
<span class="tn-subtitle">Additional Requirements for a Silver Medal:</span><br />
<ul><br />
<li><br />
Experimentally validate that at least one new BioBrick Part or Device of your own design and construction work as expected.<br /><br />
Our Best Biobrick <a href="http://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a>: the Ethylene Forming Enzyme (EFE) was shown to produce ethylene and ripen all of the fruit that we tested!<br><br />
The following parts built by us were also shown to work as expected: <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>, <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>.<br />
</li><br />
<li><br />
Document the characterization of this part in the 'Main Page' of that Part's/Device's Registry entry Submit this new part to the iGEM Parts Registry. <br />
</li> <br />
<li><br />
Describe in the wiki one or more ways in which the environment, security, safety and ethics and/or ownership and sharing or other broader implications have been taken into consideration in the design and execution of your project.<br /><br />
We have thoroughly analysed the possible pathways available in nature for ethylene production. The choice of the right pathway was done keeping always in mind safety and environmental issues. For example, we decided not to use the plant ethylene pathway, which would produce also cyanide...! Also <i>B. fruity</i> was designed with a photoinducible control system, to provide a safe alternative to commercial ripening methods that exploit ethylene. Check our <a href="https://2013.igem.org/Team:UNITN-Trento/Safety">Safety page</a> for specific info. <br />
</li><br />
</ul><br />
</div><br />
<br />
<div class="gold medal"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4a/Tn-2013Gold-medalaaa.png" /><br />
</div><br />
<div class="gold text"><br />
<span class="tn-subtitle">Additional Requirements for a Gold Medal: </span><br />
<ul><br />
<li><br />
Improve the function of an existing BioBrick Part or Device, enter this information in the 'Experience' section of that Parts's Registry entry, create a new registry page for the improved part, and submit this part to the iGEM Registry.<br /><br />
We have improved 1 existing BioBricks from the registry: <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>.<br /><br />
Our new entries for this part are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>); <br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065305">BBa_K1065305</a> (new entry for <a href=" http://parts.igem.org/Part:BBa_K952003 ">BBa_K952003</a>).<br />
</li><br />
</ul><br />
We also have resubmitted in pSB1C3 two parts from the <i>Eau de coli</i> MIT 2006 project that were discontinued. We hope that these parts will be included in the 2014 registry and will be soon available to many other teams.<br/><br />
Our entries for these parts are:<br />
<ul><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065100">BBa_K1065100</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45119">BBa_J45119</a>);<br />
</li><br />
<li><br />
<a href="http://parts.igem.org/Part:BBa_K1065103">BBa_K1065103</a> (new entry for <a href="http://parts.igem.org/Part:BBa_J45319">BBa_J45319</a>).<br />
</li><br />
</ul><br />
</li><br />
<li><br />
Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br /><br />
We have designed an innovative way to provide the consumer with ready-to-eat fruit that exploits our engineered bacterial system. For more details visit the <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">Vending Machine page</a>.<br />
</li><br />
</ul><br />
</div><br />
</div><br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Attributions</span><br />
<p><br />
We tried to work together on each aspect of the project, however each of us had a specific task to facilitate the work.<br />
</p><br />
<br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013_idea_icon.png" alt=""><br />
<span class="area"><br />
Idea<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
<i>B. fruity</i> starts from an initial idea of Caterina and was further developed and implemented by the entire team<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/d/d2/Tn-2013_cloning_icon.png" alt=""><br />
<span class="area"><br />
Cloning<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Everybody cloned at least 1 or more biobricks, however our cloning queen was Caterina.<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/e/ef/Tn-2013_eth_icon.png" alt=""><br />
<span class="area"><br />
Ethylene subteam<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Thomas with the help of Damiano Avi in the physics dept.<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/1/1a/Tn-2013_mesa_icon.png" alt=""><br />
<span class="area"><br />
MeSA subteam<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Michele, Caterina and Gabriele. Michele with the help of Marco Scapinello and Damiano Avi for characterisation by GCMS-FID.<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/0/0e/Tn-2013_blueled_icon.png" alt=""><br />
<span class="area"><br />
Blue light subteam<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Fabio, Bruno, Caterina<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/c/c0/Tn-2013_sub_icon.png" alt=""><br />
<span class="area"><br />
Bacillus subteam<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Viola, Emil<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/2/26/Tn-2013_fruit_icon.png" alt=""><br />
<span class="area"><br />
Fruit tests<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Bruno, Thomas, Michele<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/a/ac/Tn-2013_wiki_icon.png" alt=""><br />
<span class="area"><br />
Wiki<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Wiki master: Gabriele<br><br />
Graphics: Gabriele, Fabio and Thomas<br><br />
Plots: Bruno<br><br />
Contents: all team members<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/6/63/Tn-2013_icon_hp.png" alt=""><br />
<span class="area"><br />
Human Resources<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
All team members<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/8/8a/Tn-2013_vending_icon.png" alt=""><br />
<span class="area"><br />
Vending machine<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Fabio with the graphical help of Angelo Digiacomo<br />
</p><br />
</div><br />
<div class="image_wrapper"><br />
<img class="icon" src="https://static.igem.org/mediawiki/2013/9/94/Tn-2013_tshirt_icon.png" alt=""><br />
<span class="area"><br />
Logo and T-shirts<br />
</span><br />
</div><br />
<div class="text_wrapper"><br />
<p><br />
Fabio and Viola<br />
</p><br />
</div><br><br />
<span class="tn-effect">SUPPORT</span><br />
<ul><br />
<li><br />
<b>Cristina Del Bianco</b> for her unvaluable help and constant omnipresence. Our main contact for support, was able to guide us from beginning to end of the project, keeping the team united and focused on results. She also was our Instructor for an exciting Synbio/iGEM training course in the Spring!<br />
</li><br />
<li><br />
<b>Sheref Mansy</b> and <b>Olivier Jousson</b> helped us during our brainstorming and were available in the summer for consultation;<br />
</li><br />
<li><br />
<b>Paola Torre</b> helped with lab protocol procedures;<br />
</li><br />
<li><br />
<b>Rossella Tomazzolli</b>, <b>Margherita Kos</b> and <b>Matteo Grott</b> ensured a fantastic laboratory space;<br />
</li><br />
<li><br />
The laboratory of <a href="http://www.science.unitn.it/labfm/pmwiki/pmwiki.php">Atomic and Molecular Physics Laboratory</a> of the University of Trento for access to all gas chromatography equipment. <b>Damiano Avi</b> and <b>Marco Scapinello</b> for help troubleshooting the protocol for ethylene and MeSA detection;<br />
</li><br />
<li><br />
Compression tests on fruit were done in the Industrial Engineering Department (laboratory of polymers and composites, prof. Alessandro Pegoretti) under the supervision of <b>Andrea Dorigato</b>;<br />
</li><br />
<li><br />
Colorimetric assays on fruit were done in the Industrial Engineering Department (<a href="http://www.unitn.it/en/dii/28286/materials-engineering">Laboratory of Supercritical Fluids</a>, Eng. Sara Spilimbergo) under the supervision of <b>Giovanna Ferrentino</b>;<br />
</li><br />
<li><br />
Angelo Digiacomo helped us with the graphical design of B. fruity vending machine.<br />
</li><br />
</ul><br />
<br />
<span class="tn-subtitle">We would like also to thank:</span><br />
<ul><br />
<li> the University of Trento for supporting this initiative<br />
<li><br />
the <a href="http://www.unitn.it/en/cibio">Centre for Integrative Biology</a> and prof. Alessandro Quattrone for providing materials and access to instrumentation;<br />
</li><br />
<li><br />
The Mansy lab for sharing materials and protocols;<br />
</li><br />
<li><br />
prof. Graziano Guella and prof. Ines Mancini for helpful chemistry suggestions;<br />
</li><br />
<li><br />
Marzia Filippi, from the Safety office, for revising our safety measurements;<br />
</li><br />
<li><br />
LMU Munich iGEM team 2012 and prof. Mascher for sending us their <i>Bacillus subtilis</i> plasmids;<br />
</li><br />
<li><br />
The laboratory of Dr. W. Winkler at the University of Maryland and the Groeningen iGEM team 2013 for sharing Bacillus subtilis protocols;<br />
</li><br />
<li><br />
Michele Forlin for modelling advices;<br />
</li><br />
<li><br />
Michele Forlin and Fabio Chizzolini for being such great teaching assistants during the Spring.<br />
</li><br />
</ul><br />
<img id="black_board" src="https://static.igem.org/mediawiki/2013/9/91/Thank-you-blackboard_%281%29.jpg" alt="thank you all"><br />
<br />
</div><br />
<br />
<div class="sheet-2"><br />
<a href="https://2013.igem.org/Team:UNITN-Trento/Laboratory/Meetings"><br />
<img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/8/8b/Tn-2013-arr-ATTR_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/Extra/Fruit%20Info"><br />
<img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/5/53/Tn-2013-arr-ATTR_next.png" /><br />
</a><br />
</div><br />
</div><br />
<br />
<!--end content--></html>|<html>https://static.igem.org/mediawiki/2013/e/ef/Tn-2013-headerbg-Sfondogr2.jpg</html>|<html>https://static.igem.org/mediawiki/2013/5/52/Tn-2013-headerbg-Sfondogr2_OR.jpg</html>}}</div>Cridelbiancohttp://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripeningTeam:UNITN-Trento/Project/Fruit ripening2013-10-02T20:43:49Z<p>Cridelbianco: </p>
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<div class="container"><br />
<div class="sheet"><br />
<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 BBa_K1065001 for three days and then left in a jar for other five days. It is well clear that threated 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 threated 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 BBa_K651001 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 Bba_K1065001 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 BBa_K1065001 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 BBa_K651001 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 transofrmed with Bba_K1065001 in red). In the right panel Bruno show 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 BBa_K1065001 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 ing. 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 optic 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 (BBa_1065001 induced, green) and negative controls (No cells, blue and BBa_1065001 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 (BBa_1065001 induced, blue) showed a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 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 (BBa_K1065106)</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 (BBa_K1065106)</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 />
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</div><br />
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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 BBa_K1065001 for three days and then left in a jar for other five days. It is well clear that threated 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 threated 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 BBa_K651001 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 Bba_K1065001 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 BBa_K1065001 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 BBa_K651001 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 transofrmed with Bba_K1065001 in red). In the right panel Bruno show 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 BBa_K1065001 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 ing. 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 optic 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 (BBa_1065001 induced, green) and negative controls (No cells, blue and BBa_1065001 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 (BBa_1065001 induced, blue) showed a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 not induced, red).</span><br />
<br />
<span class="tn-effect">We have engineered a device able to ripen different kind of fruit!</span><br />
</div><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 (BBa_K1065106)</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 (BBa_K1065106)</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 />
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<div class="sheet"><br />
<span class="tn-title">Safety considerations</span> <br><br />
<br />
<br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<br />
<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.<br />
<li> our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities. <br><br><br />
<br />
For more detailed information read the safety considerations below. <br><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins.<br />
<font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br/><br />
<br/><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br/><br />
<br/><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. <br />
</p><br />
<br />
<p><br />
Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility for this reason.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year they are reported many accidents due to the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)</span><br />
</p><br />
<br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Working in the lab with ethylene and MeSA</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold. <br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
<br />
</ul><br />
<br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span> <br><br />
<br />
<br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<br />
<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.<br />
<li> our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities. <br><br><br />
<br />
For more detailed information read the safety considerations below. <br><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins.<br />
<font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br/><br />
<br/><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br/><br />
<br/><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. <br />
</p><br />
<br />
<p><br />
Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility for this reason.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year they are reported many accidents due to the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)</span><br />
</p><br />
<br />
<br />
<p><br />
We designed our project taking into consideration this safety aspect. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Precautions</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold. <br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
<br />
</ul><br />
<br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
</div><br />
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<div class="container"><br />
<div class="sheet"><br />
<span class="tn-title">Safety considerations</span> <br><br />
<br />
<br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<br />
<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.<br />
<li> our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities. <br><br><br />
<br />
For more detailed information read the safety considerations below. <br><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins.<br />
<font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br/><br />
<br/><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br/><br />
<br/><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene is distributed, stored and used in airtight systems always at high pressure. <br />
</p><br />
<br />
<p><br />
Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility for this reason.<br />
</p><br />
<br />
<p><br />
As described in our fruit info section, ethylene is widely used to ripen fruit before distribution to the consumers. Every year they are reported many accidents due to the explosion of ethylene cylinders in ripening facilities, or during transportation. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)</span><br />
</p><br />
<br />
<br />
<p><br />
Our system avoids these problems. Our system avoids accumulation of ethylene in cylinders, because ethylene would be produced only upon induction of our engineered bacteria. Additionally, the light sensing system that we introduced to control ethylene production, could be easily implemented in a scaled up application like the vending machine that we designed. For example, the vending machine could have ethylene detectors that controls the light and thus ethylene production. In other words, when ethylene concentrations get too high, the genetic device is shut off.<br />
</p><br />
<br />
<span class="tn-subtitle">Precautions</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold. <br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
<br />
</ul><br />
<br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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<span class="tn-title">Safety considerations</span> <br><br />
<br />
<br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<br />
<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.<br />
<li> our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities. <br><br><br />
<br />
For more detailed information read the safety considerations below. <br><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins.<br />
<font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br/><br />
<br/><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br/><br />
<br/><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<br />
</p><br />
<br />
<p><br />
Whatever the final employment, ethylene is distributed, stored and consumed in airtight systems always at high pressure. The fact that ethylene is asphyxiating, extremely flammable and explosive at 27.000 ppm renders the usage of ethylene quite dangerous. <span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility for this reason. Because of ethylene asphyxiating characteristic, the American Conference of Governmental Industrial Hygienists adopted a Threshold Limit Value of 200 ppm for ethylene as an 8-hour time-weighted average. <span class="tn-ref">(Ethylene Product Safety Assessment of <i>The Dow Chemical Company</i>, 8 Dec 2007, <a href="http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_00bf/0901b803800bfd21.pdf?filepath=productsafety/pdfs/noreg/233-00280.pdf&fromPage=GetDoc">LINK</a>)</span><br />
</p><br />
<br />
<p><br />
A very serious issue is the possibility of explosion that can cause injuries and, in some cases, death.<br />
</p><br />
<br />
<p style="padding: 0 2em;"><br />
<i>On 3 July 1987 an explosion occurred inside an ethylene oxide purification column at a chemical factory at Antwerp, Belgium. The explosion was due to decomposition of ethylene oxide. It was accompanied by a fire ball, which started a number of secondary fires. These, together with blasts and missiles, caused extensive damage. Fourteen people were injured.</i> <span class="tn-ref">(F I Khan and S. A Abbasi, <i>J LOSS PREVENT PROC</i>, 5(12):361-78, 1999)</span><br />
</p><br />
<br />
<p><br />
Even in the field of fruit ripening some cases of explosion can be found. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)</span><br />
</p><br />
<br />
<p><br />
Our system avoids these problems. In fact, our transformed strain cannot produce ethylene at a concentration high enough to be explosive. With an air/culture volume ratio equal to 4 we detected and quantified around 200 ppm of ethylene.<br />
</p><br />
<br />
<span class="tn-subtitle">Precautions</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold. <br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
<br />
</ul><br />
<br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
</p><br />
<br />
<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
<br />
<p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
</div><br />
<br />
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<div class="sheet"><br />
<span class="tn-title">Safety considerations</span> <br />
<br />
<br />
In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:<br />
<br />
<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.<br />
<li> our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities. <br><br><br />
<br />
For more detailed information read the safety considerations below. <br><br />
<br />
<span class="tn-subtitle"> Ethylene pathway selection</span><br />
<p><br />
When we decided to engineer a biological system able to produce ethylene, we looked at all the already characterized pathways. At first we checked the ethylene producing pathway from plants, because we wanted to work on fruit ripening, and we knew that ethylene is a plant hormone able to accelerate this process.<br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/><br />
<br />
<p><br />
After a careful analysis we stumbled upon a problem: the plant pathway produces hydrogen cyanide as byproduct, a highly toxic gas that inhibits cytochrome C oxidase, as well as other important proteins.<br />
<font color="red"><b>Cyanide can kill a person</b></font> within 10 minutes even at very low concentrations (around 300 ppm). Plants, however, have a detox mechanism that gets rid of this hazardous product. That's why we do not die when we eat fruit!<br />
</p><br />
<br />
<div class="tn-double-wrap"><br />
<img class="photo" title="This is the exception that proves the rule. Bruno is in fact allergic to almost all fruit and would die in any case!!!" src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-bruno_face_2.jpg" /></center><br />
<p class="text"><br />
Fruit renders hydrogen cyanide harmless thanks to the &beta;-cyanoalanine synthase enzyme, which catalyzes the synthesis of &beta;-cyanoalanine from cysteine and hydrogen cyanide. The issue is that the HCN removing reaction produces hydrogen sulfide, which is also toxic and flammable! We weren't able to find a biological way to remove the resulting hydrogen sulfide, so we searched for an alternative ethylene synthesis pathways.<br/><br />
<br/><br />
The plant pathway would have been a convenient way to produce ethylene. The pathway contains SAM synthetase, an enzyme that our team was planning to exploit for methyl salycilate production. Nevertheless, we continued looking for alternatives.<br/><br />
<br/><br />
</p><br />
</div><br />
<br />
<p><br />
We found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from <i>Pseudomonas syringae</i>. <i>Ps</i> is a plant pathogen bacteria able to produce ethylene through a reaction catalyzed by the 2-Oxoglutarate Oxygenase/Decarboxylase enzyme. This enzyme converts 2-Oxoglutarate <br />
to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>. <br />
</p><br />
<img class="no-border" src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" /><br />
<br />
<span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span><br />
<br />
<p><br />
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.<br />
</p><br />
<br />
<p><br />
Whatever the final employment, ethylene is distributed, stored and consumed in airtight systems always at high pressure. The fact that ethylene is asphyxiating, extremely flammable and explosive at 27.000 ppm renders the usage of ethylene quite dangerous. <span class="tn-ref">(Sigma-Aldrich MSDS sheet) (R A Strehlow, <i>INT SYMP ON COMB</i>, 14(1):1189-1200, 1973)</span><br />
</p><br />
<br />
<p><br />
Ethylene handling requires many precautions: the employees of every manufacturing facility must attend an advanced training program, unnecessary ethylene exposure must be limited and safety equipment have to be present in every facility for this reason. Because of ethylene asphyxiating characteristic, the American Conference of Governmental Industrial Hygienists adopted a Threshold Limit Value of 200 ppm for ethylene as an 8-hour time-weighted average. <span class="tn-ref">(Ethylene Product Safety Assessment of <i>The Dow Chemical Company</i>, 8 Dec 2007, <a href="http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_00bf/0901b803800bfd21.pdf?filepath=productsafety/pdfs/noreg/233-00280.pdf&fromPage=GetDoc">LINK</a>)</span><br />
</p><br />
<br />
<p><br />
A very serious issue is the possibility of explosion that can cause injuries and, in some cases, death.<br />
</p><br />
<br />
<p style="padding: 0 2em;"><br />
<i>On 3 July 1987 an explosion occurred inside an ethylene oxide purification column at a chemical factory at Antwerp, Belgium. The explosion was due to decomposition of ethylene oxide. It was accompanied by a fire ball, which started a number of secondary fires. These, together with blasts and missiles, caused extensive damage. Fourteen people were injured.</i> <span class="tn-ref">(F I Khan and S. A Abbasi, <i>J LOSS PREVENT PROC</i>, 5(12):361-78, 1999)</span><br />
</p><br />
<br />
<p><br />
Even in the field of fruit ripening some cases of explosion can be found. <span class="tn-ref">(<i>United Press International</i>, 20 Jan 2013, <a href="http://www.upi.com/Top_News/World-News/2013/01/20/One-killed-13-injured-in-Lebanon-blast/UPI-17131358694952/f">LINK</a>) (George Ramos, <i>Los Angeles Times</i>, 7 Jul 1999, <a href="http://articles.latimes.com/1999/jul/07/local/me-53692">LINK</a>)</span><br />
</p><br />
<br />
<p><br />
Our system avoids these problems. In fact, our transformed strain cannot produce ethylene at a concentration high enough to be explosive. With an air/culture volume ratio equal to 4 we detected and quantified around 200 ppm of ethylene.<br />
</p><br />
<br />
<span class="tn-subtitle">Precautions</span><br />
<p><br />
We carefully checked the MSDS for both <a href="http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=IT&brand=FLUKA&productNumber=03484&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/fluka/03484?lang=it&region=IT">ethylene</a> and <a href="http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=IT&language=it&productNumber=M6752&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fm6752%3Flang%3Dit">MeSA</a>. We always worked with our producing ethylene bacteria under a chemical hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed for connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. However it is commonly used as a flavouring agent and as a topical anti-infiammatory treatment. It may pose a risk when ingested at high concentrations (5 ml of MeSA have the same effect of 23 300 mg aspirin caplets) and has a lowest lethal dose of 101 mg/kg body weight in adults. Our parts characterization demonstrated that the amount of ethylene and MeSA produced by our engineered bacteria is below any dangerous reported threshold. <br />
</p><br />
<br />
<span class="tn-subtitle">Safety Form at a glance</span><br />
<p><br />
We used some strains of <i>E. coli</i> (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) and one strain of <i>B. subtilis</i> (168). All these strains belong to the risk group 1. In our project we used only one BioBrick that came from an organism of the second risk group: BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.<br />
<br />
</ul><br />
<br />
If released accidentally, our engineered microorganisms would not pose any risk to the public. The amount of ethylene or MeSA produced in the open air would be too low to be dangerous to people. The same goes for the environmental risk: both the compounds are naturally synthesized by plants. However, some issues might occur if the system is scaled-up. Our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under the control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for Trp (and also Thr, if transformed). <br />
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<span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="https://static.igem.org/mediawiki/2013/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span><br />
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<p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p><br />
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