Team:UNITN-Trento/Safety

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         <span class="tn-title">Safety form in brief</span>       
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         <span class="tn-title">Safety considerations</span>
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        <span class="tn-subtitle">
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            Ethylene pathway selection
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        </span>
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         <p>
         <p>
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             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 plants ethylene producing pathway, because we wanted to work on fruit ripening and we knew that ethylene is a plant hormone able to accelerate this process.
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            In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:
 +
            <ul>
 +
                <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>
 +
                <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>
 +
            </ul>
 +
            For more detailed information please read the safety considerations below.<br />
 +
        </p>
 +
 
 +
        <span class="tn-subtitle"> Ethylene pathway selection</span>
 +
        <p>
 +
             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.
         </p>
         </p>
         <img class="no-border" src="http://2013.igem.org/wiki/images/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/>
         <img class="no-border" src="http://2013.igem.org/wiki/images/b/bb/Tn-2013-project_ethylene-Plants_path.jpg"/>
 +
        <p>
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            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!
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        </p>
 +
        <br />
         <div class="tn-double-wrap">
         <div class="tn-double-wrap">
             <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="http://2013.igem.org/wiki/images/5/5a/Tn-2013-bruno_face_2.jpg" /></center>
             <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="http://2013.igem.org/wiki/images/5/5a/Tn-2013-bruno_face_2.jpg" /></center>
             <p class="text">
             <p class="text">
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                 But then we stumbled upon a problem in this pathway: it produces hydrogen cyanide as byproduct, an highly toxic gas that inhibits the cytochrome C oxydase enzyme.
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                 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 />
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                <font color="red"><b>It can kill a human</b></font> within 10 minutes even at very low concentration (around 300 ppm).
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                 <br />
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                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/>
+
                 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 />
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                 <br/>
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                 <br />
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                 The plant pathway would have been a convenient way to produce ethylene. It already contains SAM synthetase, that our team exploit also for methyl salycilate production. However, we tried to look further into alternative pathways.<br/>
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                 <br/>
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             </p>
             </p>
         </div>
         </div>
         <p>
         <p>
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             We found three alternative pathways: one very complicated and involving superoxide species, and the remaining two exploiting 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. It takes 2-Oxoglutarate  
+
             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  
-
             as substrate and transforms it into ethylene, water and carbon dioxyde (Goto M. Plant and Cell Physiology (2012) 26, 141-150).  
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             to ethylene, water, and carbon dioxide <span class="tn-ref">(Goto M. Plant and Cell Physiology (2012) 26, 141-150)</span>.  
         </p>
         </p>
         <img class="no-border" src="http://2013.igem.org/wiki/images/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" />
         <img class="no-border" src="http://2013.igem.org/wiki/images/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" />
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         <span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span>
         <span class="tn-subtitle">A solution to avoid unsafe storage of ethylene cylinders</span>
         <p>
         <p>
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             Ethylene is the simplest unsatured hydrocarbon. Like all hydrocarbons, it is asphyxiating and flammable. In the ripen facility it is stored in high pressure cylinders that can be very dangerous.
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             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>
         </p>
         </p>
 +
         <p>
         <p>
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            Using our system can avoid this issue. In fact, our transformed strain cannot produce ethylene in a concentration high enough to be explosive (from 2.7% to 34% v,v is needed). With an air/culture volume ratio equal to 4 we detected and quantified around 200 ppm of ethylene.
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          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.
         </p>
         </p>
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        <span class="tn-subtitle">Precautions</span>
 
         <p>
         <p>
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             We always worked with our producing ethylene bacteria under the hood. When samples were induced, cultures were maintained in special airtight vials with a rubber cap on top that allowed the connection to the micro-gas chromatograph. Also for MeSA we worked in safe conditions since it is harmful and irritating. 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>.
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             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>
         </p>
         </p>
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        <span class="subtitle">Safety Form</span>
 
         <p>
         <p>
-
             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 coming from organisms of the second risk group:
+
             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.
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            <ul>
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        </p>
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                <li>
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-
                    BBa_J45319: catalyses the production of salycilate from chorismate and comes from <i>Pseudomonas aeruginosa</i>.
+
-
                </li>
+
-
            </ul>
+
-
             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, anyway our vending machine will not contain sufficient ethylene to be risky. Besides, most of our parts are under control of inducible promoters. Furthermore the strain of <i>B. subtilis</i> that we used is auxotrophic for thriptofane (and also threonine if transformed).  
+
        <span class="tn-subtitle">Working in the lab with ethylene and MeSA</span>
 +
        <p>
 +
            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.
 +
        </p>
 +
 
 +
        <span class="tn-subtitle">Safety Form at a glance</span>
 +
        <p>
 +
            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>.
 +
        </p>
 +
        <p>
 +
             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).  
         </p>
         </p>
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         <span class="tn-effect">If you are interested in more informations and details<br />please check our <a href="http://2013.igem.org/wiki/images/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span>
+
         <span class="tn-effect">If you are interested in more informations and details<br />please check out our <a href="http://2013.igem.org/wiki/images/9/98/Tn-2013-UniTN_Trento_Safety.pdf">Safety form</a></span>
-
         <p>Safety forms were approved on September 23, 2013 by Evan Appleton.</p>
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         <p class="mini">Safety forms were approved on September 23, 2013 by Evan Appleton.</p>
     </div>
     </div>
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</div>
</div>
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<a id="tn-sp-tour" href="http://2013.igem.org/Team:UNITN-Trento/Project/Datapage">
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<a id="tn-sp-tour" href="http://2013.igem.org/Team:UNITN-Trento/Project/Datapage#tour">
     <img src="http://2013.igem.org/wiki/images/d/d7/Tn-2013-tour-F_AAA_DSC_0055.png" />
     <img src="http://2013.igem.org/wiki/images/d/d7/Tn-2013-tour-F_AAA_DSC_0055.png" />
     <span>Continue the tour!</span>
     <span>Continue the tour!</span>

Latest revision as of 22:29, 4 October 2013

Safety considerations

In our project we wanted to produce ethylene. The design and implementation of the project was done always keeping safety issues in consideration:

  • 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.
  • 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.
For more detailed information please read the safety considerations below.

Ethylene pathway selection

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.

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. Cyanide can kill a person 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!


Fruit renders hydrogen cyanide harmless thanks to the β-cyanoalanine synthase enzyme, which catalyzes the synthesis of β-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.

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.

We considered two other alternative pathways: one very complicated that involved superoxide species, and another one that exploited an enzyme derived from Pseudomonas syringae. P. syringae 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 to ethylene, water, and carbon dioxide (Goto M. Plant and Cell Physiology (2012) 26, 141-150).

A solution to avoid unsafe storage of ethylene cylinders

Ethylene is the simplest unsaturated hydrocarbon and one of the chemicals most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.(Sigma-Aldrich MSDS sheet) (R A Strehlow, INT SYMP ON COMB, 14(1):1189-1200, 1973)

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.

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. (United Press International, 20 Jan 2013, LINK) (George Ramos, Los Angeles Times, 7 Jul 1999, LINK)(FACTS - Hazardous Material Accidents Knowledge Base)

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.

Working in the lab with ethylene and MeSA

We carefully checked the MSDS for both ethylene and MeSA. 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.

Safety Form at a glance

We used some strains of E. coli (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM109) and one strain of B. subtilis (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 Pseudomonas aeruginosa.

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 B. subtilis that we used is auxotrophic for Trp (and also Thr, if transformed).

If you are interested in more informations and details
please check out our Safety form

Safety forms were approved on September 23, 2013 by Evan Appleton.

Continue the tour!