Team:UNITN-Trento/Safety

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.

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).

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 or 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 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 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.

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).

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