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From 2012.igem.org

For more insights of the safety aspects of our product in general and on the environment we invited Prof. J.D. van Elsas, head of the Microbial Ecology group of the University of Groningen. He is also a member of COGEM and expert on the environmental aspect of biosafety. He explained that the biosafety is divided in Risk Assessment and Risk Management. Risk is an equation of hazard and exposure.

Hazard is defined as the degree of harm a GM organism may cause, this depends on the nature of the insert or changed gene/operon.
Hazard may cause harm to human/animal health that come from the toxicity or allerginicity effect of the GMO. But also the environment can be exposed to harm. What is the effect of the GMO on the species diversity or does it has an effect on the functioning of the ecosystem?

Exposure is the degree to which humans/animals/the environment are exposed to the hazard introduced by the GM organism. Exposure is caused by the release of GMOs, this release can be unintended releases or a deliberate release. Once a GMO is released several thing need to be considered namely: The survivability of the GMO (establishment, colonization), gene persistence of the genetically modified gene/operon or plasmid insert via horizontal gene transfer and the effects on indigenous microbial communities.

With this knowledge we can make a risk assessment of our Food Warden system. Our final product does not contain an antibiotic resistance marker. This is a main concern for environmental safety, because the potential risk of gene transfer of an antibiotic resistance marker to a different a possibly virulent bacteria. The insert of our Food Warden bacteria consist of a promoter identified by the natural genetic response of B. subtilis on the volatiles of spoiled meat. The promoter is natural gene part taken from the genome of B. subtilis and does not contain any harmful or virulence factors.

The second part of our insert is the reporter system or pigment. We choose the gene that expresses the lycopene pigment. This pigment use in food coloring and is registered as E160d. This insert in implemented in the food grade B. subtilis natto strain. In our assessment the Food Warden bacterium does not create any hazard or risk for human, animal or environment.

Even in a worst case scenario, if our food warden bacterium is released into the environment, the plasmid will not provide an advantage for survival above the natural biodiversity. Nor could any genes potentially transferred to other bacteria give those bacteria the ability to become virulent or dominant over other bacteria, which in turn could endanger the biodiversity. The ability to produce a pigment will waste valuable energy of the bacteria. The strain we will use for our final product will also not threaten the biodiversity in the environment, because this strain a natural strain and already in the environment.

However we do not want our product to be released into the environment because not everything is known or can be ruled out. This is the risk management part of the safety aspect. In case of release in the environment we need to think about ecological containment. This is divided into three categories natural die-out of population, induced die-out of population: use of plasmid-derived toxin/antitoxin systems and safety measures: vb. steaming of soils.

We thought of the idea to use the induced die-out method. By anchoring a gene producing a toxin for B. subtilis strongly in the genome the bacteria. This gene is placed behind a strong promoter that promotes either a strong responsive stress factor or sporulation gene. Thus in the case of stress or an unfavourable environment the bacteria will expresses these genes, but instead of producing the desired pigment the toxin is produced that will kill the cell. So in the rare case the sticker is cut and the bacteria is release the environment is most likely stressful and will thus kill the cell. Also after the usage of the Food Warden system the food supply in the sticker will run out which in turn triggers the stress/sporulation gene and thereby killing the bacteria.