Team:Cornell/project/wetlab/metallothionein

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<h1 style="padding: 0px; margin-bottom: 0px;">Wetlab</h1>
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<p>Metallothioneins</p>
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<li><a href="https://2014.igem.org/Team:Cornell/project/wetlab">Overview</a></li>
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<li class="active"><a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">Metallothioneins</a></li>
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<h1 style="margin-top: 0px;">Header 1</h1>
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<h1>Construct Design</h1>
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Text text text Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt.  
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Metallothioneins are a low molecular weight, cysteine-rich family of proteins that provides protection against metal toxicity to a wide range of taxonomic groups. The thiols clustered at the core of the protein tightly chelate the metal ions by forming strong coordinate bonds.<sup>[1]</sup> Cloned and overexpressed metallothioneins can sequester metal ions transported by a metal transport system, but simultaneously inhibit growth in microorganisms. A number of metallothioneins expressed in <i>E. coli</i> had problems with stability, leading to studies conducted with stabilizing systems.<sup>[2]</sup> The system we ultimately cloned into a BioBrick was <i>crs5</i>, a gene that codes for <i>Saccharomyces cerevisiae</i> metallothionein, with a glutathione <i>S</i>-transferase carboxy-terminal fusion system (<i>GST-crs5</i>). In previous research, this fusion protein was proven to have higher stability and was approximated to be about 25% by mass of the total expressed protein of transformed <i>E. coli</i>.<sup>[3]</sup>
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Our first metallothionein BioBrick <a href="http://parts.igem.org/Part:BBa_K1460001"> (BBa_K1460001)</a> consists of <i>GST-crs5</i> synthesized with a <i>T7</i> promoter in pSC1C3. This is part of an inducible system consisting of an arabinose-activating pathway in which the araBAD promoter turns on the highly active T7 polymerase that in turn reads the metallothionein gene. Our second metallothionein BioBrick <a href="http://parts.igem.org/Part:BBa_K1460002">(BBa_K1460002)</a> consists of <i>GST-crs5</i> without the <i>T7</i> promoter for other promoters to clone into the backbone and better interweave the metallothionein’s functions with novel systems.
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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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<h1>Results</h1>
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Because successfully expressed metallothionein inhibits growth in microorganisms, we can use growth tests as a tool for determining successful expression of our metallothionein constructs. We transformed  BBa_K1460001 into <i>E.coli</i> BL21-AI and grew it and unmodified BL21-AI in LB+.1% L-Arabinose for 24 hours in an incubated plate reader at 37 degrees Celsius. Plotted below is the average OD for three biological triplicates of BL21 and BL21 BBa_K1460001. Plotted OD is corrected for OD of media.
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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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This graph displays statistically significant (student’s two-tailed t-test, p<.05) differences between unengineered BL21 and BL21 engineered to express metallothionein.  This data suggests that <i>GST-crs5</i> is being successfully expressed in this engineered strain.  Additionally, when working with these cultures for subsequent metal sequestration tests, final culture OD's were consistently observed to be less than those of wild type cells.
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When expressed, <i>GST-crs5</i> should confer resistance to heavy metal toxicity.  To test whether the construct BBa_K1460001 did in fact confer resistance to engineered cells, we grew engineered and non-engineered cells in different concentrations of mercury that we found allowed normal growth, slightly inhibited growth, and completely inhibited growth in wild type <i>E.coli</i> BL21.  These concentrations corresponded to 0.05 uM Hg, 0.5 uM Hg, and 5 uM Hg respectively.  Besides the respective heavy metal concentrations, all media contained LB and .1% L-Arabinose for induction.  For convenience, BL21 curves are graphed in pastels and BBa_K1460001 curves are graphed in dark.
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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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For concentrations of Hg that are not completely toxic to cells, we see very similar results as above for growth in no metal. Cells engineered to express metallothionein have growth inhibition when compared to wild type.  What is interesting in this experiment, however is the 5 uM concentration of Hg.  While we do see that growth is inhibited when compared to the 0.5 uM and 0.05 uM Hg concentrations for the same strain, <b>there is growth</b>.  In non-engineered BL21 there is none.  This suggests that, in fact, our construct is conferring resistance to metal toxicity to engineered cells.
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<h4>Combination with other BioBricks:</h4>
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BBa_K1460001 was combined with the BioBricks BBa_K1460003, BBa_K1460004, and BBa_K1460005 to create strains that should sequester nickel, mercury, and lead.  Results from these experiments can be found on the <a href="https://2014.igem.org/Team:Cornell/project/wetlab/nickel">nickel</a>, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/mercury">mercury</a>, and <a href="https://2014.igem.org/Team:Cornell/project/wetlab/lead">lead</a> pages. 
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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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<h1>References</h1>
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<li>Peterson, C., Narula, S., & Armitage, I. (1996). 3D solution structure of copper and silver-substituted yeast metallothioneins. FEBS Letters, 85-93.</li>
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<li>Ref 2</li>
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<li>Davis, Stephanie R., "Characterizing the role of the bacterial metallothionein, SmtA, in mammalian infection" (2011). Honors Scholar Theses. University of Connecticut. Paper 178.</li>
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<li>Ref 3</li>
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<li>Chen, S., & Wilson, D. (1997). Construction and characterization of <i>Escherichia coli</i> genetically engineered for bioremediation of Hg(2+)-contaminated environments. <i>Applied and Environmental Microbiology</i>, 63(6), 2442-2445.</li>
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Latest revision as of 03:43, 18 October 2014

Cornell iGEM

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Wet Lab

Construct Design

Metallothioneins are a low molecular weight, cysteine-rich family of proteins that provides protection against metal toxicity to a wide range of taxonomic groups. The thiols clustered at the core of the protein tightly chelate the metal ions by forming strong coordinate bonds.[1] Cloned and overexpressed metallothioneins can sequester metal ions transported by a metal transport system, but simultaneously inhibit growth in microorganisms. A number of metallothioneins expressed in E. coli had problems with stability, leading to studies conducted with stabilizing systems.[2] The system we ultimately cloned into a BioBrick was crs5, a gene that codes for Saccharomyces cerevisiae metallothionein, with a glutathione S-transferase carboxy-terminal fusion system (GST-crs5). In previous research, this fusion protein was proven to have higher stability and was approximated to be about 25% by mass of the total expressed protein of transformed E. coli.[3]

Our first metallothionein BioBrick (BBa_K1460001) consists of GST-crs5 synthesized with a T7 promoter in pSC1C3. This is part of an inducible system consisting of an arabinose-activating pathway in which the araBAD promoter turns on the highly active T7 polymerase that in turn reads the metallothionein gene. Our second metallothionein BioBrick (BBa_K1460002) consists of GST-crs5 without the T7 promoter for other promoters to clone into the backbone and better interweave the metallothionein’s functions with novel systems.

Results

Because successfully expressed metallothionein inhibits growth in microorganisms, we can use growth tests as a tool for determining successful expression of our metallothionein constructs. We transformed BBa_K1460001 into E.coli BL21-AI and grew it and unmodified BL21-AI in LB+.1% L-Arabinose for 24 hours in an incubated plate reader at 37 degrees Celsius. Plotted below is the average OD for three biological triplicates of BL21 and BL21 BBa_K1460001. Plotted OD is corrected for OD of media.
This graph displays statistically significant (student’s two-tailed t-test, p<.05) differences between unengineered BL21 and BL21 engineered to express metallothionein. This data suggests that GST-crs5 is being successfully expressed in this engineered strain. Additionally, when working with these cultures for subsequent metal sequestration tests, final culture OD's were consistently observed to be less than those of wild type cells.

When expressed, GST-crs5 should confer resistance to heavy metal toxicity. To test whether the construct BBa_K1460001 did in fact confer resistance to engineered cells, we grew engineered and non-engineered cells in different concentrations of mercury that we found allowed normal growth, slightly inhibited growth, and completely inhibited growth in wild type E.coli BL21. These concentrations corresponded to 0.05 uM Hg, 0.5 uM Hg, and 5 uM Hg respectively. Besides the respective heavy metal concentrations, all media contained LB and .1% L-Arabinose for induction. For convenience, BL21 curves are graphed in pastels and BBa_K1460001 curves are graphed in dark.
For concentrations of Hg that are not completely toxic to cells, we see very similar results as above for growth in no metal. Cells engineered to express metallothionein have growth inhibition when compared to wild type. What is interesting in this experiment, however is the 5 uM concentration of Hg. While we do see that growth is inhibited when compared to the 0.5 uM and 0.05 uM Hg concentrations for the same strain, there is growth. In non-engineered BL21 there is none. This suggests that, in fact, our construct is conferring resistance to metal toxicity to engineered cells.

Combination with other BioBricks:


BBa_K1460001 was combined with the BioBricks BBa_K1460003, BBa_K1460004, and BBa_K1460005 to create strains that should sequester nickel, mercury, and lead. Results from these experiments can be found on the nickel, mercury, and lead pages.

References


  1. Peterson, C., Narula, S., & Armitage, I. (1996). 3D solution structure of copper and silver-substituted yeast metallothioneins. FEBS Letters, 85-93.
  2. Davis, Stephanie R., "Characterizing the role of the bacterial metallothionein, SmtA, in mammalian infection" (2011). Honors Scholar Theses. University of Connecticut. Paper 178.
  3. Chen, S., & Wilson, D. (1997). Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg(2+)-contaminated environments. Applied and Environmental Microbiology, 63(6), 2442-2445.