Safety by Design

Choice of pesticide

Pesticides are toxic compounds specifically designed to interact with organism metabolisms¹. With herbicides killing undesired weeds and fungicides inhibiting fungal metabolic pathways, insecticides are focused on the reduction of crop-infested insects. Many insecticides have been reported as one of the biggest threats to honey bees by strongly compromising the functionality of their gut microbial community².

Thus, the design of a probiotic targeting this group of pesticides seems the most straightforward approach. However, we decided AGAINST it.

Introducing a genetically modified organism (GMO) into the environment already harbours many risks on its own. Uncontrollable dissemination hereby marks the biggest bottlenecks of new engineered organisms. The creation of a bacteria with high resistance against insecticides has dangerous potential to further colonise other insects targeted by these chemicals generating an even bigger threat to agriculture.

Therefore, our approach focuses on a herbicide – Glyphosate. Our engineered bacteria is very unlikely to form a symbiosis with plants targeted by glyphosate. Therefore our design would strongly reduce many of the risks related to GMO exposure and thus creates a much safer product.

Host organism

The probability of successful colonisation of probiotics rises strongly when choosing a host organism already present in the bee gut microbiome. Here, Bacillus subtilis, a non-pathogenic bacteria and GRAS organism (Generally Recognized as Safe) was selected as a protein expression host. However, Bacillus subtilis is a known spore-former. These bacterial spores can persist at extreme conditions over long periods in the environment³ and would increase the chance of undesirable uptake from other species.

We want to reduce that probability and thus the risk to the environment. Therefore, we have chosen to work with a non-spore-forming mutant (BGSC #1S129). The mutated strain was obtained from Bacillus Genetic Stock Center⁴.

Protein regulation

For high production of our recombinant protein, a strong promoter is needed. An extra control mechanism which enables protein expression only when the probiotic bacteria is proliferating inside the target host species should be considered.

In our approach, we have introduced a stationary phase promoter called P_ylb. When our probiotic colonises the bee gut microbiome, the expression of glyphosate-degrading proteins is induced only upon cells reaching the stationary phase. Glyphosate resistance will therefore not be established in species that just come into contact with our probiotic but did not reach a stable stationary phase. This design allows growth-regulated protein expression thus resulting in a safer GMO.

Laboratory Safety

To answer this far-reaching question, we were educated about safety around the lab by our supervisor, Johan Bonde. Further dangers concerning environmental exposure of our GMO were discussed with the safety department manager, Solmaz Hajizadeh and the biology safety manager, Lina Herbertsson.

For us, it was clear to follow the mantra of “Better safe than sorry”. Hereby, attention was drawn to what we called: GPSA-rule:

Gloves
Protective clothing
Safety goggles
At all times

Besides general safety measurements, the evaluation of potential risks for each experiment should help to avoid dangerous situations. Therefore, each member had to familiarise themselves with those risk assessment before performing the experiment. In addition to the safety training our team has undertaken, we want to spread further awareness of security features around the lab. A part of our Synthetic Biology Dictionary was therefore dedicated to safety. An overview of designated hazard symbols should function as a reminder of the risk they indicate and thus safety measurements required before using particular labelled chemicals. Positions of elements of protection such as fire extinguishers or defibrillators are of equal importance and are also pinpointed here The created security section within this handbook should educate future iGEMers about general safety and biosafety requirements in place to create a more secure working experience.

How can we protect the environment during laboratory work?

Scenarios - What should we do if the unplanned happens?

A team member wants to clean up the lab and finds an old, unlabelled 200 mL flask full with a clear liquid inside at the workbench. They believe it is just water and pour it into the sink. However, it turns out it was a very toxic and environmentally harmful chemical.

All wastewater coming from the sink is collected at our facility and then directed into the general sewer of Lund kommun. As it will be strongly diluted once reaching the central water system, the environmental impact might not be so severe. However, the possibility remains to stop the collected water from entering the general channel, but it is a very complex and costly process. Therefore, impact and effort need to be weighed out. Nevertheless, to prevent accidents like that, correct labelling is important. Moreover, environmental or health hazardous chemicals can be additionally labelled with pictograms. Anything carrying a potentially hazardous substance should further be stored accordingly and only used on an open bench when necessary. After usage, it should be disposed of in specifically designed waste bins or returned to its original storage unit.

The lab's centrifuge is broken. In order to harvest our GMOs, we have to walk to a different building right opposite our lab. While going over you slip on the wet flour and your flasks with GMO inside break. What do you do?

Any form of GMO exposure outside of a secure lab space needs to be reported to the University. They are specifically designed forms in a place where you can report anonymously or sign with your name. However, a supervisor, safety representative or head of the division needs to be informed immediately. It remains to take precautions to avoid these situations. Even if our GMO should not pose a risk, it is better to don’t leave flasks, tubes or similar that came into contact with GMOS unattended. When translocating high amounts of cells from one space to the other, store them in more resilient containers and use sealable transport boxes.

After iGEM, our project continues and reaches stage 2. We start to feed isolated bees with our probiotic. They are all kept in a glass cage. A new student accidentally knocks over the cage which breaks and all the bees are released into the lab.

When bringing GMOs and insects, like bees together, a three-root system needs to be put in place. By increasing the amount of barriers (e.g. doors), the possibility of breaking out will be strongly reduced⁶. Furthermore, before starting the work, one needs to assure that there is no direct way of escape. However, in case of a bee accidentally escaping, the low survival rate of isolated social bees will limit further spreading⁶. Nevertheless, the escape of individual organisms should be reported to Lund University authorities as described above.