Difference between revisions of "Team:Purdue/Genetic Pathway"

 
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<p>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.</p>
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<p>To couple the presence of a target biomarker to the activity of our reporter, we decided to use a ‘split’ version of HRP.</p>
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<p>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 <a class="ref-link" href="#">[1]</a>.</p>
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<h3>Split HRP</h3>
 
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<h3>Reconstituted HRP</h3>
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<p>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.</p>
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<p>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. </p>
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<p>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.</p>
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<th>Type of Candidiasis</th>
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<th>Vulvovaginal</th>
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<th>Systemic</th>
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<td>Yeast Phenotype</td>
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<td>Opaque</td>
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<td>White</td>
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<td>Biomarker</td>
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<td>Tyrosol</td>
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<td>Binding Protein</td>
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<p>Theoretically, split versions of these binding proteins could be used to create a binary output depending on the presence of their corresponding biomarker. </p>
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<p>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.</p>
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<p>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, <i>S. cerevisiae</i></p>
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<p>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.</p>
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<p>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.</p>
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<p>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. </p>
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<li>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</li>
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<li>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</li>
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Latest revision as of 01:24, 18 October 2018

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Genetic Construct

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

  1. 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
  2. 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

Special Thanks

to all those that made this possible

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