Emerald ash borer (EAB) infestation is a local problem in Guelph, Ontario, and throughout North America 1. In 2002, the highly invasive and foreign EAB, also known as Agrilus planipennis (A. planipennis), arrived in Canada via infested lumber2. Since then it has ravaged Canadian forests with total damages estimated to be at $1.4 billion with 99% of infested trees dying within 8-10 years of infestation3. These beetles only feast on ash trees (however, infestations of the Blue ash, Pumpkin ash, and Oregeon ash are less common) and their larvae burrow under the bark of ash trees causing conditions such as canopy thinning, structural weakening, and leaving the trees vulnerable to secondary infections5.
Current chemical and biological solutions to treat EAB infestations are limited by time, cost, rising levels of resistance in EAB, and negative environmental impact6. These methods include mechanical and silvicultural practices (cutting and burning), chemical pesticides, hybridization of ash tree species, and biological controls7.
In 2018 iGEM Missouri used Cry8Da, a cytotoxin with specificity against the EAB by binding to the receptors on the gut epithelial cells of the EAB thus causing cell lysis and death, to protect Ash trees through genetic engineering Arabidopsis thaliana (A. thaliana), a model organism8. However, the Missouri team was unsuccessful in combining their parts and were unable to develop EAB-resistant plants8. As such, no other iGEM team has successfully or otherwise used the Cry8Da protein in their project. Thus, our work is novel and aims to revolutionize EAB treatments and the ongoing battle against invasive species.
Our project focuses on utilizing a native organism Saccharomyces cerevisiae (S. cerevisiae) on Ash trees to prevent and control infestation by the highly invasive EAB. S. cerevisiae naturally inhabits the bark of ash trees9, and incorporating Cry8Da into this native species means we can protect ash trees without introducing any new chemicals or organisms into the environment and tree microbiome. By prioritizing ecosystem health our project exemplifies the Sustainable Development Goals (SDG) #1510.
Thus our biopesticide, Ash Guard, provides a greener alternative in a world that needs more sustainable options due to global warming3. EAB infestation leads to habitat destruction which causes deforestation, a tool used to prevent further spread of the EAB, which reduces ash trees and the positive impacts that they have on the environment including nutrient cycling, providing habitats for animals, and microbial biodiversity11.
Applying synthetic biology to this issue helps to avoid harmful side effects to the environment that are seen with traditional pesticides12 and combat EAB infestations. Furthermore, it exemplifies the promise of synthetic biology in areas where current science has failed to solve serious issues by creating a targeted bioinsecticide with minimal side effects. It also supports biodiversity and conserves ash tree populations13. This is accomplished by attempting to clone Cry8Da in Escherichia coli (E. coli) DH5a, Bacillus subtilis (B. subtilis), and Saccharomyces cerevisiae (S. cerevisiae). For this, we used three different plasmids: PCG004 (E. coli and B. subtilis), pET28a (E. coli), and PYES2 (S. cerevisiae).
1. Cloning Cry8Da into E. coli DH5a, B. subtilis and S. cerevisiae. 2. Testing on EAB, by spraying our cultures on the leaves collected from Ash trees. Then placing the leaves in the beetles container where they would be fed on.
For more information about our project, explore Engineering Success
The hardware project supports synthetic biology research by solving issues that all labs face - plastic waste, high costs, and equipment issues. The lab-scale Polypropylene Extruder converts plastic lab waste into high-value 3D printer filament, and the Parts Library provides a variety of low cost 3D-printable equipment accessories to make lab work more efficient and ergonomic.
Synthetic biology requires the use of significant amounts of plastic consumables such as pipette tips, tubes, petri dishes, and culture plates. To offset this waste generation and reduce disposal costs, the engineering team developed a cost-effective process for recycling used polypropylene lab waste into 3D printer filament that can be used to 3D print lab accessories in-house.
From tube holders to vortexes, synthetic biology wouldn’t be possible without this fundamental equipment. Unfortunately, common lab activities such as vortexing and uncapping/recapping microcentrifuge tubes is unergonomic and can lead to injuries over time14. Automated equipment is too expensive to be feasible for most labs15, and accessories to improve existing equipment are limited. Additionally, fundamental equipment such as tube holders are often unnecessarily bulky and inefficient due to a lack of adjustability. This is why a library of 3D printable parts, referred to as The Parts Library, was created to improve the usability and functionality of common lab equipment while reducing costs associated with upgrades. The parts are designed to facilitate and streamline lab activities by improving workflow and retrofitting equipment to make it more ergonomic. All parts are simple and adjustable to satisfy user needs and accommodate a variety of equipment models while being easy to print and use with limited 3D printing experience. Many of these parts have been used in iGEM Guelph’s lab with great success throughout this year’s project!
1. Herms, D. A., & McCullough, D. G. (2014). Emerald ash borer invasion of North America: History, biology, ecology, impacts, and management. Annual Review of Entomology, 59, 13-30. https://doi.org/10.1146/annurev-ento-011613-162051
2. Haack, R. A., Jendek, E., Liu, H., Marchant, K. R., Petrice, T. R., Poland, T. M., & Ye, H. (2002). The emerald ash borer: A new exotic pest in North America. Michigan Entomological Society, 47(3-4), 1-5.
3. Kovacs, K. F., Mercader, R. J., Haight, R. G., McCullough, D. G., & Mastro, V. C. (2010). Cost of potential emerald ash borer damage in US communities, 2009–2019. Ecological Economics, 69(3), 569-578. https://doi.org/10.1016/j.ecolecon.2009.09.004
4. Nzokou, P., Petrice, T. R., & Haack, R. A. (2006). Borate and imidacloprid treatment of ash logs infested with the emerald ash borer. Forest Products Journal, 56(6), 78-81. https://www.proquest.com/scholarly-journals/borate-imidacloprid-treatment-ash-logs-infested/docview/214646690/se-2
5. ReforestRichmond. (2020). Emerald Ash Borer (EAB) — Reforest Richmond. ReforestRichmond.org. https://www.reforestrichmond.org/emerald-ash-borer-eab
6. Nisbet, D., Kreutzweiser, D., Sibley, P., & Scarr, T. (2015). Ecological risks posed by emerald ash borer to riparian forest habitats: A review and problem formulation with management implications. Forest Ecology and Management, 358, 165-173. https://doi.org/10.1016/j.foreco.2015.08.030
7. McCullough, D. G., & Poland, T. M. (2019). Emerald ash borer (Coleoptera: Buprestidae) densities over a 6-year period on untreated trees and trees treated with systemic insecticides. Journal of Economic Entomology, 112(1), 201-211. https://academic.oup.com/jee/article-abstract/112/1/201/5106620
8. Missouri iGEM. (2018). Missouri iGEM Project. https://2018.igem.org/Team:Missouri_Rolla
9. Held, B. W., Simeto, S., Rajtar, N. N., & Cotton, A. J. (2021). Fungi associated with galleries of the emerald ash borer. Fungal Biology, 125(6), 447-458. https://doi.org/10.1016/j.funbio.2021.03.004
10. United Nations. (2015). Transforming our world: The 2030 Agenda for Sustainable Development. https://sdgs.un.org/2030agenda
11. Duan, J. J., Bauer, L. S., Van Driesche, R. G., & Gould, J. R. (2018). Progress and challenges of protecting North American ash trees from the emerald ash borer using biological control. Forests, 9(3), 142. https://doi.org/10.3390/f9030142
12. Cappaert, D., McCullough, D. G., Poland, T. M., & Siegert, N. W. (2005). Emerald ash borer in North America: A research and regulatory challenge. American Entomologist, 51(3), 152-165. https://doi.org/10.1093/ae/51.3.152
13. Gandhi, K. J. K., & Herms, D. A. (2010). North American arthropods at risk due to widespread Fraxinus mortality caused by the Alien emerald ash borer. Biological Invasions, 12, 1839-1846. https://doi.org/10.1007/s10530-009-9594-1
14. Park, J.-K., Boyer, J., & Punnett, L. (2022). Biomechanical Exposure to Upper Extremity Musculoskeletal Disorder Risk Factors in Hospital Laboratories. International Journal of Environmental Research and Public Health, 19(1), 499. https://doi.org/10.3390/ijerph19010499
15. Wenzel, T. (2023). Open hardware: From DIY trend to global transformation in access to laboratory equipment. PLoS Biology, 21(1), e3001931–e3001931. https://doi.org/10.1371/journal.pbio.3001931