Ice rinks are a cultural and recreational cornerstone in Canada, supporting over 800,000 registered skaters, hockey players, and curlers. Maintaining rink surface temperatures between -4 and -8 °C requires energy-intensive refrigeration systems, making these facilities among the largest municipal energy consumers. This study proposes transforming E. coli with modified inaZ genes, native to Pseudomonas syringae, to produce ice-nucleating proteins (INPs) that promote ice formation at higher temperatures. Our previous plasmid design used a modular cloning strategy, but this was deemed ineffective due to the high number of repeated sequences within inaZ. As such, in our current proposal, E. coli will be engineered with inaZ240 with a truncated N-terminal domain, enabling intracellular protein production. Molecular dynamics (MD) simulations of INP-water interactions demonstrated the formation of ice lattice structures, with ~600 hydrogen bonds forming over 5 μs. Experimentation on commercial INPs identified a minimum effective nucleation concentration of 0.250 g L⁻¹, capable of inducing ice formation at temperatures as high as -2°C. These results support the feasibility of INP-mediated ice nucleation as an energy-efficient alternative for ice rink maintenance.

This year, our team is continuing last year’s project, but we have made major improvements and important changes.
Our project has three main goals:
Objective 1: Genetically engineer E. coli bacteria to produce ice-nucleating proteins (proteins that help water freeze).
Objective 2: Use computer modelling to study how these proteins interact with water and determine which protein designs are most effective at triggering ice formation.
Objective 3: Educate the public about how ice rinks are maintained and work with rink operators and community members to ensure our project addresses real local needs.
Compared to last year, we significantly redesigned our experimental approach. We moved away from a modular cloning method and adopted a more traditional gene cloning strategy. This change allows us to build and test our engineered proteins in a more reliable and controlled way. We are also introducing three new modifications to improve how efficiently our proteins trigger ice formation. In addition, we will experimentally compare our engineered proteins to commercially available ice-nucleating products to evaluate their performance. Finally, we have shifted our community focus from large, high-performance facilities to local community ice rinks, where our work can have more direct and practical impact.