Team:Cornell/project/wetlab/nickel

From 2014.igem.org

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Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>,  imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup>
Because the metallothionein proteins that bind to the heavy metals are located within the <i>E. coli</i>, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein <i>nixA</i>, originating from the bacterium <i>Helicobacter pylori</i>,  imports nearby Ni<sup>2+</sup> ions into the cell.<sup>[1]</sup> Normally used by <i>H. pylori</i> to allow for urease activity,<sup>[2]</sup> <i>nixA</i> also shows promise for purposes of bioaccumulation and remediation.<sup>[3]</sup>
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The first nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460003">BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460006">BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and GST-<i>crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins.  
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The first nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460003">BBa_K1460003</a> consists of the Anderson promoter, the <i>nixA</i> gene, and a terminator, allowing for the constitutive expression of the <i>nixA</i> and the accumulation of nickel within the cell. The second nickel BioBrick <a href="http://parts.igem.org/Part:BBa_K1460006">BBaK1460006</a> was constructed by inserting our metallothionein construct, the <i>T7</i> promoter and <i>GST-crs5</i> metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of <i>nixA</i> for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins.  
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Part BBa_K1460003 in pUC57 was co-transformed with part <a href="http://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006.  To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Ni for a final nickel concentration of 1 mM.  These cultures were grown for 8 more hours.  The cells were then removed and supernatant was tested for nickel concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.   
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Part BBa_K1460003 in pUC57 was co-transformed with part <a href="http://2014.igem.org/Team:Cornell/project/wetlab/metallothionein">BBa_K1460001</a> (<i>GST-crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006.  To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Ni for a final nickel concentration of 1 mM.  These cultures were grown for 8 more hours.  The cells were then removed and supernatant was tested for nickel concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.   
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Figure 3 shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and GST-<i>crs5</i> are in fact able to remove nickel ions from water.  
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Figure 3 shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with <i>nixA</i> and <i>GST-crs5</i> are in fact able to remove nickel ions from water.  
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Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="http://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.
Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, <a href="http://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.

Revision as of 03:41, 18 October 2014

Cornell iGEM

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Wet Lab

Construct Design

Because the metallothionein proteins that bind to the heavy metals are located within the E. coli, we have constructed BioBricks containing heavy metal transport proteins that will translocate surrounding heavy metals into the cell. The high-affinity nickel transport protein nixA, originating from the bacterium Helicobacter pylori, imports nearby Ni2+ ions into the cell.[1] Normally used by H. pylori to allow for urease activity,[2] nixA also shows promise for purposes of bioaccumulation and remediation.[3]

The first nickel BioBrick BBa_K1460003 consists of the Anderson promoter, the nixA gene, and a terminator, allowing for the constitutive expression of the nixA and the accumulation of nickel within the cell. The second nickel BioBrick BBaK1460006 was constructed by inserting our metallothionein construct, the T7 promoter and GST-crs5 metallothionein gene, downstream of our first construct. This allows for the simultaneous constitutive expression of nixA for nickel uptake and accumulation and the induced expression of metallothioneins. As metallothioneins inhibit cell growth, utilizing inducible metallothionein expression permits the bacteria to adequately grow before producing metallothioneins.

BBa_K1460006

Results

Cells successfully expressing nixA should be transporting more nickel ions past the cell wall. This would lead to increased nickel sensitivity. To test for nickel sensitivity, E.coli BL21 and engineered BL21 with part BBa_K1460003 in the cmr plasmid pSB1C3 were grown for a 24 hour period in LB with 1 mM Ni.
After 24 hours of growth, no significant difference in growth was observed between the two strains. What we consistently observed as well, however, is that there is no inhibition of growth of BL21 at high concentrations of nickel (figure 2). Even if nixA is expressed and is actively transporting nickel ions into cells, it is possible that the concentration of nickel is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test nickel concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the nickel (II) chloride that we were using for testing.

Part BBa_K1460003 in pUC57 was co-transformed with part BBa_K1460001 (GST-crs5) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the nickel sequestration part BBa_K1460006. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Ni for a final nickel concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for nickel concentration using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) with the help of Cornell's Nutrient Analysis Lab. Error bars in chart represent standard deviation of three biological replicates.
Figure 3 shows the average final concentration of nickel in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460003. However, when we consider cell density and plot the amount of metal removed per OD (figure 4) there is a statistically significant difference between the two strains at a p-value of .01 (student's t-test, two-tailed). These data suggest that cells engineered with nixA and GST-crs5 are in fact able to remove nickel ions from water.

Ideally, this experiment would be run with the OD of both strains remaining the same to prevent changes in metabolite concentrations. This is difficult in this experiment because, as we have shown, cells expressing metallothionein have inhibited growth.

References


  1. Fulkerson, J., & Mobley, H. (2000). Membrane Topology of the nixA Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III. Journal of Bacteriology, 1722-1730.
  2. Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene nixA: Synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Molecular Microbiology, 97-109.
  3. 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.