Safety

Ethylene pathway selection

When we decided to engineer a biological system able to produce ethylene, we looked at all the existing natural pathways. We firstly checked the plants ethylene producing pathway.

An unwanted byproduct is produced in the last step of ethylene synthesis: hydrogen cyanide, an highly toxic gas that inhibits the cytochrome C oxydase enzyme. At very low concentration (around 300ppm) it can kill a human within 10 minutes. Plants however have a detoxyfication mechanism that gets rid of this hazardous acid. That's why we don't die when we eat a fruit!

This pathway would have been the easiest choice for us to produce ethylene because it already contains SAM synthetase that could have been exploited even for methyl salycilate production. We decided to go for a more safe alternative pathway to produce ethylene.

Pseudomonas Syrigae pv., a plant pathogen bacteria, is able to produce ethylene exploiting only one enzyme. 2-Oxoglutarate Oxygenase/Decarboxylase enzyme takes 2-Oxoglutarate as substrate and transforms it into ethylene + water + carbon-dioxyde (Goto M. Plant and Cell Physiology (2012) 26, 141-150).

A solution to avoid unsafe storage of ethylene cylinders

Ethylene is the simplest unsatured hydrocarbon. Like all hydrocarbons, ethylene is an asphyxiant and combustible. In the ripen facility it is stored in high pressure cylinders that can be very dangerous. Using our system can avoid this problem since a bacteria can not produce ethylene in a concentration high enaugh to be explosive (from 2.7% to 34% vol is needed). In fact with an air volume / culture volume ratio equal to 4, we detected and quantified about 200 ppm of ethylene. ;

Precautions taken

We always worked with our producing ethylene bacteria under the hood. When samles were induced, cultures were mainteined in special hermetially closed vials with a rubber cap that allowed the connection to the micro gas chromatograph. Also for MeSa we worked in safe conditions as it can be nocive and irritating. We carefully checked the MSDS both for ethylene and MeSa.

Safety Form

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 risk group 1. In our project we used only two biobrick coming from organisms of the second risk group that are:

• BBa_C0040: confers tetracycline resistance and comes from Acinetobacter baumannii
• BBa_J45319: catalyses the production of salycilate from chorismate and comes from Pseudomonas aeruginosa

If released accidentally, our engeneered microorganisms wouldn't 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: in fact both the compounds are produced naturally by plants. However some risk could occur in an eventual scale up of our system: however our vending machine will not contain sufficient ethylene to be of risk. Besides, most of our parts are under control of inducible promoters, furthermore the strain of B. subtilis that we used is auxotrophic for thriptofane (and also threonine if transformed). If you are interested in more informations and details please check our Safety form