Safety

At every stages of PLAnet Zero development, safety was at the forefront of our considerations both inside and outside the lab. We knew that in order to have a viable solution to plastic pollution in our environment, we needed to develop a prototype that could be safely deployed and accepted within our community.

PLAnet Zero: Safe Project Design

In our initial design, we knew that if we were to deploy a genetically modified organism that is capable of plastic degradation, containment in waste management facilities would eliminate the risks associated with environmental release. This is why we had initially decided to implement the use of bioreactors to degrade plastic waste. However, when we switched to PLA plastic degradation in composting systems, we had to once again address the issue of containment. Our team had brainstormed implementation of PLAnet Zero in compost accelerators, a commercially available mix of bacteria targeted at starting or accelerating the decomposition process that could be added in personal composting bins. However, after meeting with sustainability and composting experts, we realized that the safest implementation of our project is to limit access to only commercial and industrial in-vessel composting facilities. These facilities have to adhere to the composting regulations outlined by the Canadian Council of Ministers of the Environment (CCME) that include sterilization of the compost prior to use (CCME, 2005). Therefore, we limit the potential of environmental release through containment and sterilization by limiting our project-use to these facilities.

We took our commitment to safe project design one step further by considering additional safety features related to our chassis that would be required for full implementation. Although we are working in the laboratory setting with Escherichia coli (E. coli) to characterize our enzymes and the surface-display mechanism, we do not intend to use this in our final product. In the future, we plan to transfer our plasmid to a chassis that is normally found within compost. We have identified biosafety level 1 risk organisms such as Serratia ssp. that are abundant in the compost environment (Boulter et al., 2002 ). Furthermore, we intend to remove the antibiotic resistance genes to avoid contaminating the compost with antibiotics from the growth media and preventing horizontal transfer of antibiotic resistance genes.

Addressing Dual-Use Risk

PLAnet Zero focuses on the enzymatic degradation of PLA plastic. The ability to degrade plastics poses a risk of damaging or degrading important plastic materials in our community either through environmental release or deliberate use. The risk may seem low, as PLA plastic use is mainly limited to disposable foodware, but PLA plastic is used as a material in 3D printing, suggesting its use may grow with the popularity of 3D printing in the future. It should be noted that we are also utilizing non-canonical amino acids (ncAAs) in order to improve the enzymatic activity of our PLA-degrading enzymes. The use of ncAAs also pose a dual-use risk. In our project we demonstrated that methionine substitution for norleucine can increase binding affinity of various esterases for select substrates. While we cannot imagine a likely harmful applications of this knowledge, it a basic advance and could in principle be used to improve the properties of other any number of other enzymes.

When weighing these dual-use risks against the potential benefits of our project in the design phase, we determined that our project is still worth pursuing. Due to the other safety features implemented in our design process including containment, sterilization, and adherence to all GMO and biosafety regulations outlined by the Canadian government, we believe that the risks of misuse remain low. Our discussions with stakeholders and experts in the field suggest that PLAnet Zero has the potential to be implemented into current composting facilities across Manitoba and that it offers a real solution to the issues with PLA plastic in our community, and thus is worth pursuing to ensure diversion from landfill.

Genetic Code Engineering

The current PLAnet Zero prototype includes the expression of proteins where methionine is replaced with norleucine. Currently, the genetic code in our organisms does not include any reassigned codons and the ability to synthesize proteins with norleucine depends on the compound being present in the growth media. Further planned iterations of PLAnet Zero do not include at this time any codon reassignment or the synthetic metabolism required for endogenous ncAA production.

Genetic code engineering such as genetic expansion or genetic code reassignment is a dual-use technology. One of the major concerns is that diagnostic methods and biosecurity software implicity assume the standard genetic code is being used. Previous iGEM teams including Lethbridge 2017 and Bielefeld-CeBiTec 2017 have elaborated on the need to adjust existing methods to account for the possibility of genetic code engineering.

At this time the only possible incompatibility that PLAnet Zero has with existing bioanalytical techniques would be if mass spectrometry were used to analyze the proteins. The norleucine would be indistinguishable from leucine and isoleucine and would likely be misassigned unless the analyst was aware of the modification beforehand.

As genetic code engineering advances new procedures and technologies will likely need to be developed to account for the use of engineered genetic codes.

Ensuring Laboratory Safety

Inside the laboratory, we ensured that team members were following all safety guidelines when working on PLAnet Zero. Our wet lab team members underwent formal WHMIS and biosafety training to minimize the potential for harm within the lab. In these sessions, not only was the importance of lab coats, safety goggles, and the use of nitrile gloves emphasized, but concerns relating to biosafety hazards in the lab and the importance of safe laboratory practice when working with biological agents.We worked closely with our institutional safety officer to provide site-specific safety training with our instructors to ensure proper laboratory technique, proper-use of biological safety cabinets and disposal methods of any harmful chemicals and biological agents.

Overall, our team UManitoba members have made safety a priority at all stages of project development, to minimize harm not only to our members in the laboratory, but our community as well.

References

Canadian Council of Ministers of the Environment. (2005). Guidelines for Compost Quality. https://ccme.ca/en/res/compostgdlns_1340_e.pdf

Boulter, J. I., Trevors, J. T., & Boland, G. J. (2002). Microbial Studies of Compost: Bacterial Identification and their Potential for Turfgrass Pathogen Suppression. World Journal of Microbiology and Biotechnology. 18. 661-671. Doi: https://doi.org/10.1023/A:1016827929432