Team:Pittsburgh/Project Overview
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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 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.
Back to TopIn Vitro Protein Synthesis
We worked with our genetic circuit in vitro for two reasons. First, the paper-based sensor developed by the Collins group necessitates that the circuit works in cell-free extract. Second, and more important, protein synthesis in vitro bypasses the fickle cell. With a cell-free extract, we can add as many plasmids as we need to complete our detection circuit. Some of our constructs are also toxic to cells. Cell-free systems provide much more flexibility for the development of our sensor.
We synthesized proteins in vitro with PURExpress cell-free extract from New England BioLabs. PURExpress is a T7-mediated E. coli system. [more stuff about cell extract--Maya?]
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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:
To bypass inefficient sequestration of the substrate strand by the catalytic strand, we developed hairpin DNAzymes as illustrated above. Read more about the DNAzymes below.
The cleaved DNA strand activates a toehold switch. which mediates expression of T3 RNA polymerase:
Toehold switches are generally RNA sequences that hide the ribosome binding site in a hairpin loop, thus preventing translation. 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.
In our system, LacZ converts the yellow substrate chloramphenicol β-D-galactopyranoside into the purple β-galactosidase. Read more about the reporter below.
The expression of T3 RNA polymerase serves to amplify the original signal from the DNAzyme activity.
The concentration of metal ion can be quite low, so an amplification system will ensure a visible signal from minimal DNAzyme cleavage. Read more about the amplification system below.
DNAzyme
We worked with two DNAzymes. The thallium-specific DNAzyme was based off the work of Huang, Vazin, and Liu. The DNAzyme is coupled with erbium metal, and the susbtrate strand contains a sulfur-modified RNA base at the cleavage point. The lead-specific DNAzyme was based off the work of Lan, Furuya, and Lu. [Aife describe sequence modifications]
Back to TopToehold Switch
[Aife sequence stuff] Click here for a YouTube video illustrating the mechanism of a toehold switch.
Back to TopReporter
Hot Metal Switch signals the presence of metal ion with a color change mediated by LacZ. The substrate chloramphenicol β-D-galactopyranoside is added to the cell-free extract so that LacZ can produce β-galactosidase as soon as it's made. Because LacZ is an enzyme, the degree of color change is limited by the amount of substrate available, not the amount of enzyme produced, which is advantageous for our in vitro system. Furthermore, because the Collins toehold switches already contained lacZ, it was easier to test the rest of the system using LacZ.
We decided to use a colorimetric signal to make the sensor amenable to home use. Colors, unlike fluorescence, can be detected without additional equipment. Although an abundant amount of GFP becomes visible to the naked eye, GFP's strength lies in its fluorescence, not its color. Additionally, a cell-free system may not be able to produce enough GFP to be visible within a reasonable amount of time. Our first choice was to use the chromoprotein amilCP from the iGEM distribution kit, developed by the 2012 Uppsala Sweden team. However, sequences of the assembled plasmid suggested we had CFP, not amilCP. No color was observed in vitro or in the cells.
Back to TopAmplifier
The amplification system was inspired by the work of Wang, Barahona, and Buck.
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