Difference between revisions of "Team:Pittsburgh"

 
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<h2>Project Description</h2>
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<p>We are developing a cell-free sensor that conditionally expresses a chromoprotein only in the presence of thallium. The metal ion detection is based on a DNAzyme that is cleaved in the presence of thallium. The released DNA strand activates a toehold switch regulated by the T7 promoter system, which will promote transcription of T3 and T7 RNA polymerases. The transcription of additional T7 RNA polymerase will amplify the signal from thallium. The T3 RNA polymerase will activate the expression of a chromoprotein. Our circuit will also incorporate a differential amplifier to remove noise from other heavy metals such as mercury. For the differential amplifier, a second DNAzyme with a greater affinity for other metals will be cleaved and produce a DNA strand complementary to that from the thallium DNAzyme. This will remove noise from other heavy metals that may also cleave the thallium DNAzyme.</p>
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<p>Early in the brainstorming process, our team wanted to develop a biosensor, since previous projects that hijacked glucometers and pregnancy tests sparked our interest. In deciding what to detect, we looked for analytes that were less widely studied and eventually decided upon thallium. Thallium, like lead and mercury, its neighbors on the periodic table, is toxic to humans. Because it has a similar atomic radius as potassium, thallium follows potassium pathways in the body. Thallium poisoning in low dosages results in hair loss and damage to the peripheral nervous system. In high doses, thallium is lethal. Thus, detection of thallium is important, especially in areas with industries that use thallium.</p>
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<p>We are also looking for collaborators. If you are interested, please contact us at pitt.igem.2016@gmail.com. We look forward to an exciting summer!
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    <h1 style="text-align:center;">HOT METAL SWITCH</h1>
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        <p style="font-size:170%; line-height:1.3 em; text-align:center;"><img src="https://static.igem.org/mediawiki/2016/a/ab/T--Pittsburgh--homeMedal.gif" style="width:22px;padding:0;"> Gold Medal &nbsp;&nbsp;&nbsp;&nbsp; <img src="https://static.igem.org/mediawiki/2016/8/85/T--Pittsburgh--homeSprout.jpg" style="width:30px;padding:0;">Nominated for Best Environment Project</p>
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     <h3 style="text-align:center; font-style:italic;">a metal detection system</h3>
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    <h2>THE PROBLEM</h2>
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    <p>Thallium is the element between lead and mercury on the periodic table. It is a byproduct of glassmaking, electronics factories, drug production, and the extraction of metals from ore (<a href="#ref">EPA</a>). Like its periodic table neighbors, thallium is a heavy metal that is highly toxic to humans--in fact, it is more potent than lead. While the EPA’s maximum contaminant level (MCL) for lead is 0.015 milligrams per liter, the MCL for thallium is only 0.002 milligrams per liter. Thallium is also known as the “poisoner’s poison” because it takes several days to kill, is odorless, and is tasteless. Thallium owes much of its toxicity to its size, which is similar to that of potassium, so thallium can easily enter cells via potassium pathways (<a href="#ref">RSC</a>). In low doses, thallium causes hair loss and problems with the kidney, liver, and intestines, which can lead to vomiting and diarrhea (<a href="#ref">ATSDR</a>, <a href="#ref">EPA</a>).</p>
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     <p>Although thallium poisoning from drinking water does not pose a huge problem in most areas of the world, thallium’s high toxicity merits a powerful detection system. Current monitoring methods require extensive preparation and lab equipment, and they are not accessible to the general population (<a href="#ref">NEMI</a>). Thus, we aim to develop an inexpensive, simple system that people can use to check for thallium in their drinking water.</p>
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     <p style="font-size:150%; line-height:1.3 em;">Hot Metal Switch is a cell-free sensor that conditionally expresses <i>lacZ</i> only in the presence of the target metal, either lead or thallium. It is made of two main parts: a cell-free extract, which contains the transcriptional and translational enzymes necessary to produce proteins from DNA or RNA <i>in vitro</i>, and a genetic circuit, which detects the metal and produces a signal. The metal ion detection is based on specific DNAzymes that are cleaved in the presence of their target metal. The released DNA strand activates a toehold switch regulated by the T7 promoter system, which will promote transcription of T3 RNA polymerase. The T3 RNA polymerase will activate the expression of <i>lacZ</i>, which will produce a color change visible to the naked eye. The T3 RNA polymerase will also activate additional expression of itself to amplify the original signal from the DNAzyme cleavage.</p>
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    <h2>THE SOLUTION</h2>
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    <p>In 2014, <a href="#ref">Pardee <i>et al.</i></a> developed a paper-based detection system that was cheap, easy to use, and efficient, producing results in less than an hour. Their system relied on RNA-based sensors to sense small molecules. These sensors were freeze-dried onto paper with cell extract, which contains the translational and transcriptional machinery a cell uses to express proteins. We are adapting their system to sense metals by using a DNAzyme-based sensor. Read more about our solution in our <a href="2016.igem.org/Team:Pittsburgh/Project_Overview" target="blank"> Project Overview</a>.</p>
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    <p style="font-size:150%; line-height:1.3 em;">The project is largely inspired by <a href="http://www.cell.com/abstract/S0092-8674(14)01291-4" target="_blank">"Paper-Based Synthetic Gene Networks"</a> from the Collins group, which details a paper-based sensor made by freeze-drying cell-free extract and the sensor onto paper. The cell-free extract is activated by rehydrating the paper with water. The paper sensor can be stored in a refrigerator. The Collins group added RNA-based sensors to detect mRNA, and in the presence of the target mRNA, the paper sensor produced a fluorescent or colorimetric response in less than an hour. Hot Metal Switch interfaces the sensor module with a genetic reporter to generate a metal-sensing circuit. Thus, it expands the system developed by the Collins lab to detect metals.</p><br>
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<!--<p>We are developing a cell-free sensor that conditionally expresses a chromoprotein only in the presence of thallium. The metal ion detection is based on a DNAzyme that is cleaved in the presence of thallium. The released DNA strand activates a toehold switch regulated by the T7 promoter system, which will promote transcription of T3 and T7 RNA polymerases. The transcription of additional T7 RNA polymerase will amplify the signal from thallium. The T3 RNA polymerase will activate the expression of a chromoprotein. Our circuit will also incorporate a differential amplifier to remove noise from other heavy metals such as mercury. For the differential amplifier, a second DNAzyme with a greater affinity for other metals will be cleaved and produce a DNA strand complementary to that from the thallium DNAzyme. This will remove noise from other heavy metals that may also cleave the thallium DNAzyme.</p>
  
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<p>Early in the brainstorming process, our team wanted to develop a biosensor, since previous projects that hijacked glucometers and pregnancy tests sparked our interest. In deciding what to detect, we looked for analytes that were less widely studied and eventually decided upon thallium. Thallium, like lead and mercury, its neighbors on the periodic table, is toxic to humans. Because it has a similar atomic radius as potassium, thallium follows potassium pathways in the body. Thallium poisoning in low dosages results in hair loss and damage to the peripheral nervous system. In high doses, thallium is lethal. Thus, detection of thallium is important, especially in areas with industries that use thallium.</p>
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    <h4>References</h4>
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    <p>Environmental Protection Agency (EPA). <a href="https://www.epa.gov/ground-water-and-drinking-water/table-regulated-drinking-water-contaminants" target="_blank">Table of Regulated Drinking Water Contaminants</a>. 2016, July 15.</p>
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<p>We are also looking for collaborators. If you are interested, please contact us at pitt.igem.2016@gmail.com. We look forward to an exciting summer!
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    <p>Royal Society of Chemistry (RSC). <a href="http://www.rsc.org/chemistryworld/podcast/Interactive_Periodic_Table_Transcripts/Thallium.asp" target="_blank"> Chemistry in Its Element - Thallium</a>. </p>
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     <p><a href="https://www.nemi.gov/home/" target="_blank"> National Environmental Methods Index (NEMI)</a>.</p>
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Latest revision as of 20:16, 5 November 2016

Thank you to our sponsors!

Office of the Provost Department of Bioengineering Department of Biological Sciences Department of Chemistry Swanson School of Engineering
IDT NEB Experiment

Gold Medal      Nominated for Best Environment Project

Hot Metal Switch is a cell-free sensor that conditionally expresses lacZ only in the presence of the target metal, either lead or thallium. It is made of two main parts: a cell-free extract, which contains the transcriptional and translational enzymes necessary to produce proteins from DNA or RNA in vitro, and a genetic circuit, which detects the metal and produces a signal. The metal ion detection is based on specific DNAzymes that are cleaved in the presence of their target metal. The released DNA strand activates a toehold switch regulated by the T7 promoter system, which will promote transcription of T3 RNA polymerase. The T3 RNA polymerase will activate the expression of lacZ, which will produce a color change visible to the naked eye. The T3 RNA polymerase will also activate additional expression of itself to amplify the original signal from the DNAzyme cleavage.

The project is largely inspired by "Paper-Based Synthetic Gene Networks" from the Collins group, which details a paper-based sensor made by freeze-drying cell-free extract and the sensor onto paper. The cell-free extract is activated by rehydrating the paper with water. The paper sensor can be stored in a refrigerator. The Collins group added RNA-based sensors to detect mRNA, and in the presence of the target mRNA, the paper sensor produced a fluorescent or colorimetric response in less than an hour. Hot Metal Switch interfaces the sensor module with a genetic reporter to generate a metal-sensing circuit. Thus, it expands the system developed by the Collins lab to detect metals.