iGEM Stockholm 2018 Project Description
Background and problem
Water is a vital natural resource, indispensable for the preservation of life on Earth. Over the last decades, however, it has suffered an alarming rate of deterioration, mainly as a result of human industrial activity. One of the most impactful polluters of water resources are pharmaceutically active substances such as antibiotics. Specifically, sulfamethoxazole (SMX) is among the most reported pharmaceuticals found in the Baltic Sea , which surrounds our home city of Stockholm. SMX is used to treat common bacterial infections, but has an ecotoxic effect on the environment.
We believe this problem is worth tackling by our team for two reasons: (1) the environmental importance of maintaining high quality sources of clean water in our community and (2) the health concern of exposing non-pathogenic bacteria to permanent low doses of antibiotics which may contribute to the soaring of antibiotic resistance.
We want to tackle this issue by producing an antibiotic degrading enzyme and investigate its capability to inactivate SMX and possibly other antibiotic compounds. Using enzymatic treatment for water bioremediation has shown to be a promising approach due to its efficacy and reduced energy need. Thus, we propose the use of laccases, a family of oxidoreductases oxidizing a wide range of aromatic compounds. Recent studies have demonstrated its capacity to eliminate pharmaceuticals such as antibiotics .
Laccases are found in a wide variety of organisms. The redox potential of these enzymes depends on the organism of origin, where laccase produced by Trametes versicolor has shown to have the highest redox potential . For this reason, we aim to use immobilized laccase from T. versicolor to inactivate the biological activity of antibiotic contaminants in water.
We have chosen Pichia pastoris and Escherichia coli as our main expression systems of the wild type laccase. Additionally, we plan to run a toxicity assay to ensure that the inactivation of the chosen antibiotics does not increase their ecotoxic properties.
One of the unique aspects of our project is the implementation of rational enzyme design. We are making sure that our recombinant enzyme folds properly even after adding the extensive α-factor sequence and His-tag, by using molecular dynamics simulations (MD). The amino acid sequence will also be mutated in a computer, with the goal of optimizing (A) the docking of the substrate to the active site, and (B) the activation energy of the reaction. Our final outcome will be an improved enzyme that will have less promiscuity and higher specificity for its substrate.
Education, Engagement and Integration
Our strategy in engaging and educating different sectors of the local and general population is to use pedagogical and interactive media. We use these means to raise awareness about the rising levels of medications in aquatic environments and the global problem of antibiotic resistance. Additionally, we intend to consult with experts and organizations working locally with the environment and wastewater, to discuss possible applications of our solution and to address safety and sustainability aspects.
 HELCOM. “Pharmaceuticals in the aquatic environment of the Baltic Sea region - A status report”, 2017.
 J. Schwarz, M. O. Aust, and S. Thiele-Bruhn, “Metabolites from fungal laccase catalysed transformation of sulfonamides” Chemosphere, vol. 81, pp. 1469– 1476, 2010.
 L. Lloret, G. Eibes, T. A. Lú-Chau, M. T. Moreira, G. Feijoo, and J. M. Lema, “Laccase-catalyzed degradation of anti-inflammatories and estrogens” Biochem. Eng. J., vol. 51, pp. 124–131, 2010.