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    <div class = "container"> <h1 style="text-align:center; font-size: 75px;"><font face= "Poiret One">Project Design</font></h1>
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              The goal of Aptapaper is to take a protein biomarker found in a patient sample and convert it into a detectable signal. Our design consists of three main steps: proximity dependent ligation, nucleic acid sequence based amplification (NASBA), and cell-free expression. Proximity dependent ligation works by using two single-stranded DNA molecules with aptamer ends. These aptamer ends are single-stranded DNA molecules that are engineered to bind to either side of our target protein. Once the aptamers seek out and bind to our target protein, the single-stranded DNA molecules are brought into very close proximity. A DNA bridge, which is complementary to sequences on both single-stranded DNA molecules, binds to the two strands, holding them in place. T4 DNA ligase then ligates the two long DNA molecules so that they form one continuous DNA strand. This DNA strand encodes the alpha-fragment of the lacZ gene. The alpha fragment is used rather than the full lacZ gene to limit the length of the single stranded DNA probes; extremely long probes have not been tested in proximity dependent ligation reactions to our knowledge. In order to drive transcription of the lacZ alpha-fragment, one of the single-stranded DNA molecules contains a T7 promoter. The DNA is double-stranded in the promoter region, a requirement for T7 polymerase activity.</font></p>
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<p><hr><img src="https://static.igem.org/mediawiki/2016/6/65/Proximity-Dependent_Ligation_Diagram.png" width=100%; height=400px;><<hr></p>         
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The lacZ alpha fragment is then transcribed, producing mRNA. This mRNA must be amplified by NASBA to detect lower amounts of protein biomarkers in patient samples. NASBA uses Avian Myeloblastosis Virus Reverse Transcriptase to reverse transcribe DNA from the mRNA template. Note, the primers used for reverse transcription contain T7 promoters so that the final double-stranded product is transcriptionally active. We then must free the DNA from the RNA:DNA hybrids, in order to produce double stranded DNA. To do this, we use RNAse H, which degrades the RNA in the RNA:DNA hybrids.  AMV reverse transcriptase is then able to fill in the single-stranded DNA, making it double-stranded. The double-stranded DNA contains a T7 promoter, enabling it to express the lacZ alpha-fragment. It is important to note that this reaction can be performed at a constant temperature near 37oC so it does not require specialized lab equipment.</font></p>
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The alpha fragment that is produced complements with the lacZ delta M15 mutant, which is constitutively expressed in the same reaction mixture on a separate DNA template. When the two fragments come together, they form the functional lacZ enzyme, which is able to break down X-gal, producing a colorimetric output. Our entire system could be freeze dried onto paper, allowing it to be stored for long periods of time at room temperature. </font></p>
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<div class = "container"> <h1 style="text-align:center;"><font face ="Poiret One">Design</font></h1>
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<p>Tuberculosis (TB) is one of the leading causes of death worldwide according the World Health Organization (source:http://www.who.int/gho/tb/en/), despite the fact that it is curable and treatments are often completely paid for by the government. Even so, 1.4 million died from TB in 2014 (source:http://www.who.int/mediacentre/factsheets/fs104/en/). Access to treatment isn't the problem. The problem is patients with TB are not being diagnosed until it is too late, since current methods are either cheap but only 50% sensitive, or are accurate but, prohibitively expensive or require access to advanced medical facilities.</p>
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                <p>Our team decided to tackle this problem by developing a genetic switch that could detect TB biomarkers with less hassle than ELIZA and other similar methods of protein detection. In the regions where TB is most prevalent, namely africa and sub-continental asia, access to labs and lab equipment is difficult. Our genetic switch is designed to be incorporated into a paper-based test strip that could be easily self administered for screening purposes with no specialized equipment. </p>
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                <p>Our switch design this year relies on two unique aptamers that both bind to the target protein. For the purpose of our experiments, this target was thrombin because it is readily available and has been thoroughly characterized. However, we anticipate converting the target protein to a TB biomarker such as CFP-10 which is highlighted in "IFN-γ/TNF-α ratio in response to immuno proteomically identified human T-cell antigens of Mycobacterium tuberculosis - The most suitable surrogate biomarker for latent TB infection." (Prabhavathi et al.) would not be very difficult. These two aptamers have been designed with sequences that enable the free-floating tails to ligate with each other through proximity dependent ligation. When the aptamers bind to the target protein and ligate together, they allow expression of the lacZ alpha fragment, which enables free floating lacZ to convert Xgal to 5,5'-dibromo-4,4'-dichloro-indigo and cause a blue colorimetric output. </p>
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                <p>When this system is freeze dried onto the paper test strip it can last up to a year with no refrigeration (Pardee, Green, et al.). To use, a consumer need only rehydrate with a sputum sample and watch for the color to change.</p>
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Latest revision as of 02:13, 20 October 2016

Home Project Modeling Team BioBrick Human Practices Safety Awards

Project Design

The goal of Aptapaper is to take a protein biomarker found in a patient sample and convert it into a detectable signal. Our design consists of three main steps: proximity dependent ligation, nucleic acid sequence based amplification (NASBA), and cell-free expression. Proximity dependent ligation works by using two single-stranded DNA molecules with aptamer ends. These aptamer ends are single-stranded DNA molecules that are engineered to bind to either side of our target protein. Once the aptamers seek out and bind to our target protein, the single-stranded DNA molecules are brought into very close proximity. A DNA bridge, which is complementary to sequences on both single-stranded DNA molecules, binds to the two strands, holding them in place. T4 DNA ligase then ligates the two long DNA molecules so that they form one continuous DNA strand. This DNA strand encodes the alpha-fragment of the lacZ gene. The alpha fragment is used rather than the full lacZ gene to limit the length of the single stranded DNA probes; extremely long probes have not been tested in proximity dependent ligation reactions to our knowledge. In order to drive transcription of the lacZ alpha-fragment, one of the single-stranded DNA molecules contains a T7 promoter. The DNA is double-stranded in the promoter region, a requirement for T7 polymerase activity.

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The lacZ alpha fragment is then transcribed, producing mRNA. This mRNA must be amplified by NASBA to detect lower amounts of protein biomarkers in patient samples. NASBA uses Avian Myeloblastosis Virus Reverse Transcriptase to reverse transcribe DNA from the mRNA template. Note, the primers used for reverse transcription contain T7 promoters so that the final double-stranded product is transcriptionally active. We then must free the DNA from the RNA:DNA hybrids, in order to produce double stranded DNA. To do this, we use RNAse H, which degrades the RNA in the RNA:DNA hybrids. AMV reverse transcriptase is then able to fill in the single-stranded DNA, making it double-stranded. The double-stranded DNA contains a T7 promoter, enabling it to express the lacZ alpha-fragment. It is important to note that this reaction can be performed at a constant temperature near 37oC so it does not require specialized lab equipment.

The alpha fragment that is produced complements with the lacZ delta M15 mutant, which is constitutively expressed in the same reaction mixture on a separate DNA template. When the two fragments come together, they form the functional lacZ enzyme, which is able to break down X-gal, producing a colorimetric output. Our entire system could be freeze dried onto paper, allowing it to be stored for long periods of time at room temperature.