Safety is undoubtedly an important part of the iGEM competition. As our current pandemic crisis taught us, taking biosafety into strict consideration should always be a milestone in laboratory procedures daily, to avoid outbreaks or unpredicted side effects. As iGEMers, we, iGEM team UNILausanne 2021, were deeply concerned about biosafety issues as well as willing to follow good lab practices and developed our project by integrating safety principles since the beginning of our experimental design.
This year, the iGEM UNILausanne team was welcomed in one of the University of Lausanne’s laboratories for 2 months and then our PI, Prof. Yolanda Schaerli, welcomed our team in her lab located in the Department of Microbiology of the University of Lausanne. To classify its laboratories, The University of Lausanne follows the ordinance on the use of organisms in a confined environment (OUC, RS 814.92) established by the Swiss Confederation. The labs we used this year are biosafety level 1 (BSL1).
Before starting any experiments one of our instructor, Dr. Audam Chhun, organized a week of bootcamp for our team during which we were taught how to properly and safely use all the equipment at our disposal in addition to the correct disposing of particular chemical wastes that could damage the environment if not well eliminated. We were also fortunate to receive training with Mr. Patrick Michaux who works for the security, environment, prevention service at our university and with whom we discussed the risks that could occur in a lab and how to avoid them.
Therefore, in order to practice in the lab, each member was required to :
- Wear a lab coat every time they enter the lab.
- Use gloves when working with microorganisms.
- Eyewear when using toxic products such as mitomycin C.
- Closed-toes footwear and long-legged trousers need to be worn.
- Wash and sanitize their hands periodically.
- Wear a facial mask to limit the spread of the SARS-CoV-2 virus.
Last, but not least, to prevent contamination working on sterile conditions as much as possible was encouraged. Besides, at the end of the day, the benches were cleaned with disinfecting solutions.
Equipment and Product use
The equipment and product used were mostly not harmful. However, one can never be too careful and good lab practices were always applied. Our team members worked with a transilluminator that has an UV lamp to see the bands on gels. UV may cause health issues if exposed. We had to be extremely careful when we had to use the light and had to make sure that the lamp was turned off before opening the sliding door that gave us access to the platform on which we had to put the gel on.
Some of our bacteria possessed plasmids with gene resistance to antibiotics; mainly they had a resistance to kanamycin, ampicillin, gentamycine or chloramphenicol. Those antibiotics are safe for human beings, nevertheless, there are side effects if they are ingested in high quantities. Thus, it was still imperative to stay vigilant when using them.
Last, mitomycin C was a product that required eyewear and gloves when handled because it is cancerigenic.
Information on Microorganisms
The objective of Aprifreeze was to use different microorganisms to fight our main target Pseudomonas syringae, that causes damage to apricot trees’ leaves. One of our subprojects consists of using a phagemid and a helper phage, which is a plasmid-like entity that gathers all the genes needed for phage's construction. Once found in the same bacterial cell they enable the formation of viral particles to achieve the deletion of a specific gene in P. syringae. The phagemid used in our project was purchased on Addgene and we ordered the helper phage at the Yale Coli Genetic Stock Center : they are classified as BSL1, thus it was possible for us to work with them in our lab while staying cautious.
Then, as explained on our website, tailocins evolved from phages and are secreted by bacteria to kill their target strains. The range targeted by tailocins is specific to a narrow range of strains. We choose to extract tailocins from P. syringae DSM50252 and
Finally, the extraction of AFP expressed in Escherichia Coli (BSL1) was performed: many different strains were used during our lab work such as E. coli BL21, E. coli NEB5α, E. coli pLys. Our instructors kindly gave us those strains to work on.
Antifreeze proteins (AFP) are produced by various organisms living in extremely cold temperatures. These proteins bind to ice crystals and inhibit their growth, which protects the organisms producing them from damage.
Throughout our project, we conducted several interviews to consult experts in a variety of topics. To discuss safety issues and to better understand the legalities of GMO production, we met with Anne-Gabrielle Wüst-Saucy and Sylvain Aubrey, two professionals working for the Swiss Federal Office for the Environment and for Agriculture respectively (link for more information).
From this discussion, we learned that AFPs are considered to be bioactive molecules and would therefore be classified as a phytosanitary product. When using these products, it is important to consider various specifications and measures, such as the purity of the final solution, its toxicity and specificity, and so on. It is also essential to evaluate any danger it could have on humans, animals, the environment, and biodiversity. Various tests need to be done, first in the lab, and then in the environment.
For our project, the sequence of the AFPs was not changed and the proteins we produced are identical to the ones naturally present in the cold-temperature living organisms from which they originate. They are therefore not considered GMOs. Nevertheless, we used modified E. coli cells to synthesize the AFPs - it is important to make sure that no trace of this bacteria can be found in the final solution. Amongst other things, it is essential that the product does not contaminate the environment, whether it be during the testing phase, or during transportation.
The goal of this part was to first extract tailocins from P. syringae DSM50252 and test them on P. syringae B301D: according to the activity spectrum on which tailocins are active and the fact that they do not carry any genetic material, they are not considered harmful. The only potential encountered was the use of mitomycin C which can induce severe damages to our health, thus strict protection gear was needed such as lab coats, gloves and goggles. Moreover, it was critical to work on sterile conditions during the whole duration of the process to restrain contamination of our samples and our environment.
In designing our project, we took special care to minimize as much as possible risks and adverse effects our product could have on the environment. We therefore decided to use tailocins, as their narrow killing range reduces the likelihood of adverse effects on beneficial bacteria species, compared to other toxins, such as broad spectrum antibiotics for example. Moreover, in contrast to phages, tailocins are not able to replicate autonomously and can therefore not persist long in the environment. Thus, they allow us to take advantage of both the specificity and potent killing ability of phages, without raising the safety and ethical questions of releasing engineered or non native phages in the environment.
As tailocins do not contain any genomic information and cannot replicate autonomously, they are like the AFPs considered as a bioactive molecule in the Swiss law and must follow the same homologation process. Their possible off-target effect should be evaluated, by checking in the lab their effect on plants and soil microbiome. To avoid damage to the environment, experiments in the lab should be conducted to demonstrate that they do not target beneficial bacteria before any field test. As for the AFPs, we plan to produce the tailocins using a GMO, once again the production process should ensure that no trace of the producing bacteria remains in the final product.
The phagemid can be maintained into the bacterial cell without killing it, as being unable to replicate nor producing new viruses. The reason for this is that the helper phage is not present in the target bacteria (i.e. Pseudomonas syringae) and thus it will not allow the phagemid to be encapsulated in new virions as they will simply not be generated. This will prevent the infection of other bacterial entities, lowering the risks of its employment. While constructing the engineered plasmid (i.e. proof of Concept of phagemid usage) the use of sodium hydroxide (NaOH) was required. NaOH is known to be a corrosive product on metals and the skin : it was then mandatory to wear gloves and goggles.