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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. 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. All assays were conducted with both a lac inducible promoter and a constitutive promoter.
Photobleaching Results from September 4, 2014
Photobleaching Results from October 15, 2014
References:
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
Superoxide Generators with WT Lac Promoter
Superoxide Generators with Constitutive Promoters
