Team:UNITN-Trento/Safety

From 2013.igem.org

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         <span class="tn-title">Safety considerations</span>       <br>
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         <span class="tn-title">Safety considerations</span>
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        <p>
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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:
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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:
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            <ul>
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                <li>we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.</li>
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<li> we decided not to use the plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.
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                <li>our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities.</li>
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<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>
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            </ul>
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            For more detailed information read the safety considerations below.<br />
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For more detailed information read the safety considerations below. <br>
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        </p>
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          <span class="tn-subtitle"> Ethylene pathway selection</span>
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        <span class="tn-subtitle"> Ethylene pathway selection</span>
         <p>
         <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.
             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.
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         <p>
         <p>
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             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.
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             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!
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            <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>
         </p>
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             <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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                 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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                 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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                 <br/>
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                 <br />
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                 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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                 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 />
             </p>
             </p>
         </div>
         </div>
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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>
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         <p>
         <p>
             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>
             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>
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         <p>
         <p>
             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.
             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.
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</p>
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        </p>
         <span class="tn-subtitle">Working in the lab with ethylene and MeSA</span>
         <span class="tn-subtitle">Working in the lab with ethylene and MeSA</span>
         <p>
         <p>
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             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.  
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             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.
         </p>
         </p>
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         <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 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>.
             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>.
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        </p>
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            </ul>
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        <p>
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             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).  
             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>

Revision as of 21:25, 1 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 plant ethylene pathway, which also produce cyanide, and we selected a more safe alternative pathway.
  • our system is inducible and avoids the accumulation of ethylene, thus providing a safer alternative to the storage of ethylene in ripening facilities.
For more detailed information 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 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. 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 found three alternative pathways: one was very complicated and involved superoxide species, and the remaining two exploited the same enzyme derived from Pseudomonas syringae. Ps 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 chemical 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: 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.

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. (United Press International, 20 Jan 2013, LINK) (George Ramos, Los Angeles Times, 7 Jul 1999, LINK)

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.

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 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.

Safety Form at a glance

We used some strains of E. coli (NEB 10beta, NEB5 alpha, BL21, TOP10, TB1, JM29) 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: 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.

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