Difference between revisions of "Team:Pittsburgh/Proof"

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<div style="max-width:1000px; margin:0 auto; padding:0px 10px 10px 10px;"> 
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    <p>Our proposed mechanism works!</p></div>
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    <p>Our proposed circuit to sense heavy metals involves several parts, as described in the <a href="/Team:Pittsburgh/Project_Overview" target="_blank">Project Overview</a>.</p>
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    <img src="https://static.igem.org/mediawiki/2016/8/8d/T--Pittsburgh--Proof_circuit.jpg" style="display:block; float:right; width:40%"><p>First, the presence of the target metal allows the DNAzyme to cleave its substrate. The cleavage product then activates the toehold switch, which produces T3 RNA polymerase. The T3 then translates the reporter protein and activates an amplification system.</p>
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    <p style="clear:both;">Our experiments show that the metal detection section of the circuit works as expected; that is, the presence of a metal causes DNAzyme cleavage, and the cleavage product activates the toehold switch to produce LacZ. In a PURExpress <i>in vitro</i> protein synthesis reaction that contains 5 ng/μL of the toehold switch DNA, 3.74 nM of the hairpin DNAzyme, 2 μM Pb(CH<sub>3</sub>CO<sub>2</sub>)<sub>2</sub> · 3H<sub>2</sub>O, and the remaining volume in water, LacZ expression is higher than the background. This can be seen with the naked eye, as shown in the image below:</p>
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    <img src="https://static.igem.org/mediawiki/2016/e/e9/T--Pittsburgh--Proof_plate.jpg" style="display:block; float:left; width:50%; padding:0 5px 5px 0px;">
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        <td>3.47 μM lead hairpin with 2 μM lead in lead buffer</td>
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        <td>3.47 μM lead hairpin with 2 μM lead in lead buffer</td>
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        <td>3.47 μM lead hairpin</td>
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        <td>3.47 μM lead hairpin</td>
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        <td>No hairpin</td>
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        <td>No hairpin</td>
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        <td>3.47 nM lead hairpin</td>
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        <td>3.47 nM lead hairpin</td>
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        <td>3.47 nM DNA trigger</td>
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        <td>3.47 nM DNA trigger</td>
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        <td>3.47 μM lead hairpin with 2 μM lead in water</td>
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        <td>3.47 μM lead hairpin with 2 μM lead in water</td>
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        <td>3.47 nM DNA trigger</td>
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        <td>3.47 nM DNA trigger</td>
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        <td>3.47 nM lead hairpin with 2 μM lead in water</td>
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        <td>3.47 nM lead hairpin with 2 μM lead in water</td>
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        <td>3.47 nM lead hairpin with 2 μM lead in buffer</td>
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        <td>3.47 nM lead hairpin with 2 μM lead in buffer</td>
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    <p style="clear:both;">The difference is also evident in an absorbance measurement. The absorbance was read at 570 nm for the figure below.</p>
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    <img src="https://static.igem.org/mediawiki/2016/3/3b/T--Pittsburgh--Results_LeadHp.jpg" style="display:block; margin:auto; width:70%;">
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    <p>Our circuit was able to detect a lead concentration of 2 μM, or about 414 parts per billion (ppb). The maximum contaminant level (MCL) set by the EPA is 72.4 nM, which is 15 ppb. However, lead levels in Flint were found to be at 13,200 ppb (<a href="http://www.npr.org/sections/thetwo-way/2016/04/20/465545378/lead-laced-water-in-flint-a-step-by-step-look-at-the-makings-of-a-crisis" target="_blank">NPR</a>). The detection limit of our sensor is definitely below this level. In addition, the <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3071848/" target="_blank">original DNAzyme</a> off which we based the hairpin structure had a detection limit of 3.7 nM. Although we did not have the chance to test our sensor over a range of lead concentrations, it has the potential to detect lead at concentrations in contaminated water.</p>
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Latest revision as of 00:49, 20 October 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

Our proposed mechanism works!

Our proposed circuit to sense heavy metals involves several parts, as described in the Project Overview.

First, the presence of the target metal allows the DNAzyme to cleave its substrate. The cleavage product then activates the toehold switch, which produces T3 RNA polymerase. The T3 then translates the reporter protein and activates an amplification system.

Our experiments show that the metal detection section of the circuit works as expected; that is, the presence of a metal causes DNAzyme cleavage, and the cleavage product activates the toehold switch to produce LacZ. In a PURExpress in vitro protein synthesis reaction that contains 5 ng/μL of the toehold switch DNA, 3.74 nM of the hairpin DNAzyme, 2 μM Pb(CH3CO2)2 · 3H2O, and the remaining volume in water, LacZ expression is higher than the background. This can be seen with the naked eye, as shown in the image below:

3.47 μM lead hairpin with 2 μM lead in lead buffer 3.47 μM lead hairpin with 2 μM lead in lead buffer 3.47 μM lead hairpin 3.47 μM lead hairpin No hairpin No hairpin
3.47 nM lead hairpin 3.47 nM lead hairpin 3.47 nM DNA trigger 3.47 nM DNA trigger 3.47 μM lead hairpin with 2 μM lead in water 3.47 μM lead hairpin with 2 μM lead in water
3.47 nM DNA trigger 3.47 nM DNA trigger 3.47 nM lead hairpin with 2 μM lead in water 3.47 nM lead hairpin with 2 μM lead in water 3.47 nM lead hairpin with 2 μM lead in buffer 3.47 nM lead hairpin with 2 μM lead in buffer

The difference is also evident in an absorbance measurement. The absorbance was read at 570 nm for the figure below.

Our circuit was able to detect a lead concentration of 2 μM, or about 414 parts per billion (ppb). The maximum contaminant level (MCL) set by the EPA is 72.4 nM, which is 15 ppb. However, lead levels in Flint were found to be at 13,200 ppb (NPR). The detection limit of our sensor is definitely below this level. In addition, the original DNAzyme off which we based the hairpin structure had a detection limit of 3.7 nM. Although we did not have the chance to test our sensor over a range of lead concentrations, it has the potential to detect lead at concentrations in contaminated water.