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Revision as of 10:22, 17 October 2018
Introduction
Idea
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. 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 their 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. By attaching the split fractions of HRP to split fractions of proteins which bind to the biomarkers indicative of C. albicans, it should be possible to equate the functionality of HRP to the presence or absence of a yeast infection.
We have identified from literature the molecules tyrosol and farnesol as biomarkers representative of invasive and vulvovaginal candidiasis, respectively. Likewise, tyrosinase and pqsR are proteins which bind tyrosol and farnesol, respectively. The generation and selection of these split proteins which compose the “locking mechanism” was our primary cloning focus.
Although one of the papers we referenced extensively, xxxx, used rational design to achieve a similar goal, we lacked the time and expertise in protein engineering necessary to confidently gauge which split-sites would be best to test. Thus, we decided to generate a varied library of potential split proteins by utilizing circular permutations.
It is hard to describe the exact process process we used without first explicitly describing the desired outcome: a library of plasmids containing two operons, each capable of generating the complementary halves of a split protein of interest bound to complementary halves of split HRP.
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