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

 
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h1> Background Information </h1>
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<p> There are two major techniques we apply in our experiment design.
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One of them is the usage of split proteins. These are proteins whose coding regions have been split in half in thier DNA in order to create a binary effect by producing 2 different protein halves. These are used to allow protein-protein interactions. In our case, we have our sensing protein being split that is also bound to a split reporter molecule, in our case it will be HRP. According to <a href="#">...'s</a> research, HRP was split in various locations to serve as a lock mechanism. However, the ideal spot to split the protein wasn't known at the time. Instead, they did a process called rational design where they analyzed the protein to find sites where this process would most likely succeed. They then created different variations where they tested their mechanism at these various sites.  
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Using their findings, we are using the 58 site for the HRP split. </p>
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However, unlike what the paper did, we didn't/don't know if our locking mechanism will work. So to do this, we needed a way to find how to split our sensing protein at sites that worked for our purposes. Since, most of our team lacks the training and the background involving protein structure and mechanics. Therefore, we are doing irrational design. This is a process where we generate various different split sequences. The procedure we used is similar to what is found in this paper <a href:"">here</a>
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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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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</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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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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<h3>Split HRP</h3>
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<h3>Reconstituted HRP</h3>
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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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<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>Farnesol</td>
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<td>Binding Protein</td>
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<td>Tyrosinase</td>
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<td>pqsR</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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<h1>References</h1>
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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

Bootstrap Example

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

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