Team:Cornell/project/wetlab/reporters

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<h1>Construct Design</h1>
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The sequestration of our targeted heavy metals by metallothioneins has a noticeable rate change when approaching saturation.<sup>[1]</sup> Therefore, a saturation detection system for each of the three metals, composed of a reporter downstream from a heavy metal inducible promoter, was proposed. The expression of the reporter, <i>amilCP</i>, would then negatively correlate to the amount of heavy metals present in the cells and thus the amount that the metallothioneins had failed to sequester. Two BioBricks for the metallothionein saturation detection system were designed: <i>amilCP</i> was inserted behind a nickel/cobalt activated promoter, P<i>rcn</i> <a href="http://parts.igem.org/Part:BBa_K1460009">(BBa_K1460009)</a>, and behind a mercury activated promoter, P<i>merT</i> <a href="http://parts.igem.org/Part:BBa_K1460010">(BBa_K1460010)</a>.  
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These reporter systems would be used in tandem with sequestering strains.  Cultures of these cells would be placed downstream of cultures of sequestering strains in a continuous system.  Once the metallothioneins in the upstream culture became saturated with heavy metals (so they can no longer take any metal out of solution), metal concentrations entering the reporter cultures would be higher.  We could, therefore, continuously monitor concentrations of heavy metals leaving our filtration system by detecting colorimetric changes in these reporter cultures.
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Dry lab's filtration box is designed to incorporate the metallothionein saturation system. The reporter constructs would be placed into a second hollow fiber reactor after the first. If the water contains metal ions after passing through the fiber reactor with the metallothioneins, <i>amilCP</i> will be expressed and turn blue. It would then be possible to find the relationship between concentrations of heavy metal in the water and the cultures’ color gradient. This could be used to indicate that the metallothioneins are saturated and the sequestration cultures need to be replaced.
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Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean. A small river named Duden flows by their place and supplies it with the necessary regelialia.
 
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But I must explain to you how all this mistaken idea of denouncing pleasure and praising pain was born and I will give you a complete account of the system, and expound the actual teachings of the great explorer of the truth, the master-builder of human happiness.
 
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, vex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
 
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No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it? But who has any right to find fault with a man who chooses to enjoy a pleasure that has no annoying consequences, or one who avoids a pain that produces no resultant pleasure? On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee.
 
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The quick, brown fox jumps over a lazy dog. DJs flock by when MTV ax quiz prog. Junk MTV quiz graced by fox whelps. Bawds jog, flick quartz, ex nymphs. Waltz, bad nymph, for quick jigs vex! Fox nymphs grab quick-jived waltz. Brick quiz whangs jumpy veldt fox. Bright vixens jump; dozy fowl quack.
 
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<li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation. <i>Applied and Environmental Microbiology</i>, 5383-5386.</li>
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Latest revision as of 03:53, 18 October 2014

Cornell iGEM

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Wet Lab

Construct Design

The sequestration of our targeted heavy metals by metallothioneins has a noticeable rate change when approaching saturation.[1] Therefore, a saturation detection system for each of the three metals, composed of a reporter downstream from a heavy metal inducible promoter, was proposed. The expression of the reporter, amilCP, would then negatively correlate to the amount of heavy metals present in the cells and thus the amount that the metallothioneins had failed to sequester. Two BioBricks for the metallothionein saturation detection system were designed: amilCP was inserted behind a nickel/cobalt activated promoter, Prcn (BBa_K1460009), and behind a mercury activated promoter, PmerT (BBa_K1460010).

These reporter systems would be used in tandem with sequestering strains. Cultures of these cells would be placed downstream of cultures of sequestering strains in a continuous system. Once the metallothioneins in the upstream culture became saturated with heavy metals (so they can no longer take any metal out of solution), metal concentrations entering the reporter cultures would be higher. We could, therefore, continuously monitor concentrations of heavy metals leaving our filtration system by detecting colorimetric changes in these reporter cultures.

Dry lab's filtration box is designed to incorporate the metallothionein saturation system. The reporter constructs would be placed into a second hollow fiber reactor after the first. If the water contains metal ions after passing through the fiber reactor with the metallothioneins, amilCP will be expressed and turn blue. It would then be possible to find the relationship between concentrations of heavy metal in the water and the cultures’ color gradient. This could be used to indicate that the metallothioneins are saturated and the sequestration cultures need to be replaced.

References

  1. Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation. Applied and Environmental Microbiology, 5383-5386.

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