Difference between revisions of "Team:Pittsburgh"
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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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<p>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 small molecules, and in the presence of the molecules, the paper sensor produced a fluorescent or colorimetric response in less than an hour. Hot Metal Switch replaces the RNA sensors with the a genetic circuit that detects metal ions. Thus, it expands the system developed by the Collins lab to detect metals.</p><br> | <p>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 small molecules, and in the presence of the molecules, the paper sensor produced a fluorescent or colorimetric response in less than an hour. Hot Metal Switch replaces the RNA sensors with the a genetic circuit that detects metal ions. Thus, it expands the system developed by the Collins lab to detect metals.</p><br> | ||
| − | <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> | + | <!--<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> |
<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> | <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> | ||
<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! | <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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Revision as of 01:18, 16 October 2016
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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 small molecules, and in the presence of the molecules, the paper sensor produced a fluorescent or colorimetric response in less than an hour. Hot Metal Switch replaces the RNA sensors with the a genetic circuit that detects metal ions. Thus, it expands the system developed by the Collins lab to detect metals.
