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<span class="tn-title">Safety considerations</span>
<span class="tn-title">Safety considerations</span> <br>
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.
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!
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).
Ethylene is the simplest unsaturated hydrocarbon and one of the chemical most used worldwide. Like all hydrocarbons, it is asphyxiating and flammable.
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. (Sigma-Aldrich MSDS sheet) (R A Strehlow, INT SYMP ON COMB, 14(1):1189-1200, 1973)
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. (Ethylene Product Safety Assessment of The Dow Chemical Company, 8 Dec 2007, LINK)
A very serious issue is the possibility of explosion that can cause injuries and, in some cases, death.
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. (F I Khan and S. A Abbasi, J LOSS PREVENT PROC, 5(12):361-78, 1999)
Even in the field of fruit ripening some cases of explosion can be found. (United Press International, 20 Jan 2013, LINK) (George Ramos, Los Angeles Times, 7 Jul 1999, LINK)
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.
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.
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.