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Team:Carnegie Mellon/Superoxide
From 2014.igem.org
Superoxide Generator Improvement
iGEM Registry of Standard Biological Parts, Part:BBa K1184000
In 2013, the Carnegie Mellon iGEM team engineered an alternative to antibiotics through the use of phage therapy and the superoxide generator known as KillerRed. The light-activated production of ROS by KillerRed kills bacteria. In 2014, we worked to further characterize the effects of KillerRed using several methods(Source: Carnegie Mellon iGEM 2013)
Methods Used
- Optimization of codons in KillerRed by eliminating rare codon usage.
- Optimization of codons in SuperNova, the monomeric version of KillerRed (Takemoto et al. 2013), by eliminating rare codon usage.
- Photobleaching analysis of KillerRed, Codon Optimized KillerRed, and Codon Optimized Supernova.
Photobleaching Chamber:
Results
From the overnight cultures, the codon optimized versions of the KillerRed and SuperNova were observed to be significantly redder in color (see photo here) . Photobleaching analysis of codon optimized KillerRed and codon optimized SuperNova in comparison to the original KillerRed, we found that the codon optimized versions showed a significant decrease in fluorescence in response to photobleaching. Viability assays also showed a significant decrease in viability for the codon optimized versions of KillerRed, compared to the viability of the original KillerRed.
Resources:
Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T. 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3:2629. doi: 10.1038/srep02629
