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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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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. 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. 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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<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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Revision as of 00:15, 12 October 2018

Bootstrap Example

Genetic Construct

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.

Special Thanks

to all those that made this possible

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Contact Us

igem@purdue.edu

Kevin Fitzgerald, B025

1203 W State St.

West Lafayette, IN 47906