Team:Purdue/Genetic Pathway
Horseradish peroxidase (HRP) is an enzyme frequently used for its ability to oxidize colorless compounds and form colored products. Tetramethylbenzidine (TMB), one such colorless compound, reacts with HRP in the presence of hydrogen peroxide to form a blue product (TMB’). Due to its speed and distinct color change, this colorimetric reaction is often used as the visible output or reporter in paper-based diagnostic tests.
To couple the presence of a target biomarker to the activity of our reporter, we decided to use a ‘split’ version of HRP.
By separately transcribing and translating amino acids 1-58 and 59-308 composing HRP into respective peptide chains, the protein can be split into a binary switch of sorts: functional HRP can only be formed when both constituent parts recombine. Martell et. al, 2016 engineered a split version of HRP which does not spontaneously reconstitute at room temperature [1].
Split HRP
Reconstituted HRP
We have additionally identified from literature the molecules tyrosol and farnesol as biomarkers indicative of invasive and vulvovaginal candidiasis, respectively. Likewise, tyrosinase and pqsR are proteins which bind tyrosol and farnesol, respectively.
| Type of Candidiasis | Vulvovaginal | Systemic |
|---|---|---|
| Yeast Phenotype | Opaque | White |
| Biomarker | Tyrosol | Farnesol |
| Binding Protein | Tyrosinase | pqsR |
Theoretically, split versions of these binding proteins could be used to create a binary output depending on the presence of their corresponding biomarker.
By linking these split binding proteins to split HRP proteins, the presence of yeast can be directly linked to the presence or absence of a blue input.
To develop a test capable of detecting C. albicans, we first had to assemble and transform a gene expressing wild-type HRP into expression chassis, S. cerevisiae
References
- Martell, J., Yamagata, M., Deerinck, T., Phan, S., Kwa, C., & Ellisman, M. et al. (2016). A split horseradish peroxidase for the detection of intercellular protein–protein interactions and sensitive visualization of synapses. Nature Biotechnology, 34(7), 774-780. doi: 10.1038/nbt.3563
- Tafelmeyer, P., Johnsson, N., & Johnsson, K. (2004). Transforming a (β/α)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution. Chemistry & Biology, 11(5), 681-689. doi: 10.1016/j.chembiol.2004.02.026
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