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From 2012.igem.org

For more insights into the safety aspects of our product in general and its possible effects 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 (see our 'in the lab' safety page for a description) and an expert on the environmental aspect of biosafety. He explained that biosafety is divided between risk assessment and risk management. Risk is often difined as a multiplication of hazard and exposure.

Hazard

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. Hazardous effects to humans or other organisms can be divided in two types, namely the toxicity or allerginicity effects of the GMO. In addition to humans and animals, the environment can be exposed to harm. It is important to consider what the effect of the GMO is on ecological diversity or whether it has an effect on the functioning of the ecosystem.

Exposure

Exposure is the degree to which humans/animals/the environment come into contact with a hazard introduced by the GM organism. Exposure is caused by the release of GMOs. This release can be unintended or deliberate. Once a GMO is released, several things must be considered, including 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 goal is to create a device that 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. However, the backbone biobrick we created is a insertion vector, thereby minimizing the chance of transferring the insert into other organisms significantly.

The insert of our Food Warden bacteria consists of a promoter identified by the natural genetic response of B. subtilis to the volatiles released during meat spoilage. The promoter is a natural gene part taken from the genome of B. subtilis and does not contain any harmful/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 a registered food additive (E160d) in the European Union.

We now use the harmless bacterium Bacillus subtilis as our chassis, but in the future we want to implement our insert in the food grade B. subtilis natto strain to make sure that the possible harm is even further minimized.

Concluding, in our assessment the Food Warden bacterium does not create any hazard or risk to humans, animals or the environment.

But imagine that...

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 biodiversity. The ability to produce a pigment will waste valuable energy within the bacteria, further lessening the strains survivability. Because the strain we use is naturally abundant, it will very unlikely cause shifts in the biodiversity.

...The reality: prevention

Despite the precautions outlined above, the product is still not intended for release into the environment, since 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, for instance the 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 a strongly responsive stress factor. Thus, in the case of stress/an unfavourable environment the bacteria will express these genes, producing the toxin that will kill the cell. So in the rare case that the sealing of the sticker is compromised and the bacteria is released, the most likely stressful environment will induce self destruction of 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.

Another, very simple method to sterilize the bacteria after usage, would be the introduction of a third, breakable compartment inside our sticker. This compartment should contain a bacteriotoxic compound and a strong color, clearly different from the pigment. In this way, a consumer can kill the bacteria directly after use by breaking the compartment and releasing the toxin inside the sticker. One big drawback however is that the safety of this completely relies on the consumer: when he or she forgets to use the third compartment, the bacteria will stay alive. This is why a genetic system might be preferred.