Team:Cornell/project/wetlab/metallothionein

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<h1>Construct Design</h1>
<h1>Construct Design</h1>
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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 (GST-<i>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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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 GST-<i>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 GST-<i>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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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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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-<i>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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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, GST-<i>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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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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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.