Laboratory Safety
Safety is a substantial part of any biological experiment and environment. Following intensive training performed by Patrick Michaux, in charge of the security, environment, prevention service at the University of Lausanne, safety measures were applied by each member of the team. They included wearing appropriate Personal Protective Equipment (PPE) such as laboratory coats, gloves and closed shoes. We also ensured that we had tied hair when appropriate, and long pants that covered legs entirely. In addition to the dress code, no food nor drinks were allowed in our laboratory. In specific cases, other preventive measures were applied to ensure individual safety such as earmuffs and eyewear. In addition, the application of the following guidelines was respected:
- No visitors were allowed in laboratory.
- Hands were washed after handling biological material, after removing gloves and when leaving the laboratory.
- All procedures were performed carefully to minimize the creation of splashes or aerosols.
- Contaminated liquids or solid materials were decontaminated before disposal (i.e. by autoclaving).
- Bottle carriers, carts or other secondary containers were used when transporting chemicals through hallways or between buildings. The individual transporting the chemical knew about its associated hazards and knew how to handle a potential spill of the material.
- The location of emergency equipment and how to use them, including safety showers, eyewash stations, medical cabinets and fire extinguishers was known by each member following a safety training which took place during a bootcamp week organized and delivered by Dr. Audam Chhun and Emanuele Boni, two supervisors of the team.
University of Lausanne follows the Swiss Confederation ordinance on the use of organisms in a confined environment (OUC, RS 814.92) to classify its laboratories. Two laboratories from the University were lent to our team. Both were classified as biosafety level 1 (BSL1), corresponding to Standard Microbiological Laboratories. Biological materials were manipulated on open benches or under chemical fume hood when handling hazardous chemical agents. Benches were cleaned with 70% ethanol after usage.
Chemical use: Most of the chemicals used throughout our project were not toxic to human beings. Nevertheless, we sometimes used hazardous chemicals, such as SybrSafe (a nucleic acid gel stain). It is safer than traditional DNA intercalating agents, but it might still have carcinogenic effects, so we handled it with extra care.
Safety Project Design
Our project aimed to fight the quagga invasion with the employment of two substances: FitD toxin and zosteric acid. We engineered bacteria in order to produce these substances. In the framework of our project, we wanted to ensure that these bacteria and substances could not harm us or the environment.
FitD toxin
We chose the FitD toxin (FitD protein), naturally produced by P. protegens CHA0, as a mussel control agent because it has already been studied as an insecticide (Péchy-Tarr et al., 2013) and researchers have shown the toxicity of a related strain (CL145A) against Dreissena mussels (Molloy et al., 2013 (A)).
Based on existing literature, we could assume that the fit toxin is heat-labile and starts rapidly degrading after 24h in water (Molloy et al., 2013 (A)). Furthermore, publications suggest that the toxins activity is highly selective to Dreissena mussels (Molloy et al., 2013 (B)). All together, these properties of the FitD toxin are sufficient to rule out the risk of possible poisoning of other creatures in case our product should be used in real life.
CHA0 inactivation
To produce the FitD toxin, we used the bacterium P. protegens CHA0, a well-characterized strain studied in the department of fundamental microbiology on our campus. We increased its natural Fit toxin production by transforming it with plasmids to overexpress the FitD toxin (Design ).
The FitD toxin is not released into the environment by P. protegens, but remains an outer-membrane protein in the cell wall. Therefore we could not easily separate the toxin from the genetically modified bacteria. This posed a problem, since the Swiss regulation currently prevents the release of genetically modified organisms into the environment. Therefore, we decided to inactivate the bacteria after they produced a large amount of FitD toxin. Bead-beating proved to be a very effective inactivation method (Experiments). It is a mechanical way of killing bacteria by letting them collide with ceramic bills in a machine that stirs the sample (bead-beater). Bead-beating allowed the inactivation of P. protegens without adversely affecting the protein (Proof of concept).
By inactivating our bacteria, we obtained a solution containing lysed cells and our toxin. In real-life application of this solution, simple adjustments of the inactivation procedure would ensure that no living cells are released in the environment.
Within the framework of the project, our product was tested exclusively in the laboratory on mussels, following standard laboratory rules to prevent contamination or infection due to bacteria and hinder any release of bacteria, quagga or larvae into the environment (Measurement).
Zosteric Acid
To produce zosteric acid, we used the bacterium E. coli BL21 (DE3) which we transformed with plasmids designed for two cooperative purposes: first, to enhance the uptake of a precursor compound (sulfate); second, to overexpress the enzymes that constitute the zosteric acid synthesis pathway (Design).
As with FitD, zosteric acid should also be applicable in infested pipes and other critical water supply systems. Once again, to ensure we do not release genetically modified bacteria in the environment, we designed our project as follows: we decided to separate the zosteric acid from the genetically modified bacteria, after sufficient production. The separation was performed by centrifugation, yielding pellets consisting of the modified bacteria which were disposed of properly (e.g autoclave) while all further experiments were carried out with the bacteria-free supernatant.
As for the fitD constructs, the zosteric acid production was tested exclusively in the laboratory, following standard laboratory rules to prevent contamination or infection with bacteria and hinder any release of bacteria into the environment (Safety Lab Work).
Quagga larvae
After receiving confirmation that experiments on mussels are allowed in Switzerland, we tested our bacteria on live mussels in the laboratory. We therefore had to ensure that no mussel larvae would enter the sewage system and cause blockages in the future. We considered all water and laboratory materials that had been in contact with the quagga mussels to be contaminated. After usage, petri-dishes and any other labware were discarded as contaminated materials and not cleaned at the sink. The mussel water was also disposed of correctly as contaminated liquid.
References
- (A) Daniel P. Molloy, Denise A.Mayer, Michael J.Gaylo, John T.Morse,Kathleen T.Presti, Paul M.Sawyko, Alexander Y.Karatayev, Lyubov E.Burlakova,Franck Laruelle, Kimi C.Nishikawa, Barbara H.Griffin. (May 2013 ). Pseudomonas fluorescens strain CL145A - a biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae). Journal of Invertebrate Pathology. https://pubmed.ncbi.nlm.nih.gov/23295683/
- (B) Daniel P. Molloy, Denise A.Mayer, Laure Giamberini, Michael J.Gaylo, (9. January 2013). Mode of action of Pseudomonas fluorescens strain CL145A, a lethal control agent of dreissenid mussels (Bivalvia: Dreissenidae). Journal of Invertebrate Pathology. https://doi.org/10.1016/j.jip.2012.12.013
- Maria Péchy-Tarr, Naomi Borel, Peter Kupferschmied, Vincent Turner, Olivier Binggeli, Dragica Radovanovic, Monika Maurhofer and Christoph Keel, Naomi Borel, Peter Kupferschmied, Vincent Turner, Olivier Binggeli, Christoph Keel, Dragica Radovanovic. (2013). Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad. Environmental Microbiology (2013) 15(3), 736–750. https://doi.org/10.1111/1462-2920.12050