Team:Pittsburgh/Project Overview
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Learn how Hot Metal Switch works!
Contents
Hot Metal Switch
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 expresses proteins from DNA in vitro and allows the sensor to work, 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.
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In Vitro Protein Synthesis
We synthesized proteins in vitro with PURExpress cell-free extract from New England BioLabs. PURExpress contains T7 transcriptional proteins. [more stuff about cell extract]
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Genetic Circuit
As described in the introductory section, the genetic circuit contains four components. When the target metal is present, a metal-specific DNAzyme is activated, resulting in cleavage of a second DNA strand:
Read more about the DNAzyme below.
The cleaved DNA strand activates a toehold switch. which mediates expression of T3 RNA polymerase:
Read more about the toehold switch below.
The T3 RNA polymerase transcribes two genes. The expression of lacZ results in a color change, which alerts the user that the target metal is present.
Read more about the reporter below.
The expression of T3 RNA polymerase serves to amplify the original signal from the DNAzyme activity.
Read more about the amplification system below.
DNAzyme
Back to TopToehold Switch
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