Team:Cornell/project/wetlab

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<h1 style="margin-top: 0px;">Idea</h1>
<h1 style="margin-top: 0px;">Idea</h1>
Mercury, nickel, and lead were targeted for sequestration by our strain of bacteria by utilizing the efficient binding properties of the pea metallothionein and specificity of the respective metal transport proteins, merT/merP<sup>1</sup>, nixA<sup>2</sup>,and CBP4. First, the yeast metallothionein<sup>3</sup> as well as each of the three heavy metal transport proteins were biobricked. Then, our wetlab sub team combined the parts to develop functional composite constructs. The idea of utilizing of metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this idea has never been investigated for lead primarily due to a lack of characterization of the lead transporter.
Mercury, nickel, and lead were targeted for sequestration by our strain of bacteria by utilizing the efficient binding properties of the pea metallothionein and specificity of the respective metal transport proteins, merT/merP<sup>1</sup>, nixA<sup>2</sup>,and CBP4. First, the yeast metallothionein<sup>3</sup> as well as each of the three heavy metal transport proteins were biobricked. Then, our wetlab sub team combined the parts to develop functional composite constructs. The idea of utilizing of metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this idea has never been investigated for lead primarily due to a lack of characterization of the lead transporter.
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  To test the sequestration efficiency of each metal, E.coli BL21-A1 was transformed with the pea metallothionein as well the respective metal transporter for the targeted metal.  
  To test the sequestration efficiency of each metal, E.coli BL21-A1 was transformed with the pea metallothionein as well the respective metal transporter for the targeted metal.  
The sequestering strains can be placed into fiber reactors to develop functional sequestering filters as part of the dry lab portion of our project.
The sequestering strains can be placed into fiber reactors to develop functional sequestering filters as part of the dry lab portion of our project.
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Revision as of 02:30, 16 October 2014

Cornell iGEM

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Wet Lab



Idea

Mercury, nickel, and lead were targeted for sequestration by our strain of bacteria by utilizing the efficient binding properties of the pea metallothionein and specificity of the respective metal transport proteins, merT/merP1, nixA2,and CBP4. First, the yeast metallothionein3 as well as each of the three heavy metal transport proteins were biobricked. Then, our wetlab sub team combined the parts to develop functional composite constructs. The idea of utilizing of metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this idea has never been investigated for lead primarily due to a lack of characterization of the lead transporter.
To test the sequestration efficiency of each metal, E.coli BL21-A1 was transformed with the pea metallothionein as well the respective metal transporter for the targeted metal. The sequestering strains can be placed into fiber reactors to develop functional sequestering filters as part of the dry lab portion of our project.
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Components

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Experiments

The growth rates of the sequestering strain were measured using spectrophotometry. In addition, two methods were used to determine heavy metal sequestration efficiency.

  1. A spectrophotometer to analyze and compare the kinetic growth rates of E. coli cultures expressing either the metallothionein, transporter proteins, both metallothionein and transporter, and just the vector backbone, as a control.
    • We hypothesized that the control culture would be mildly sensitive to growth in metal-containing media, while the cultures with the transport protein would be more sensitive to growth in metal-containing media due to increased access of the heavy metal to cellular machinery. Finally, bacteria transformed with both the metal transporter and metallothionein protein would be the least sensitive in metal-containing media. Each strain was grown in different heavy metal concentrations.
  2. Sequestration efficiency was measured by growing both, the wild type strains as well as sequestering strains in heavy metals of different concentrations. The concentration of heavy metals after growth was measured in two ways:
    • Nutrient Analysis Lab at Cornell University
    • Using green-fluorescent heavy metal indicator Phen Green

Results

  1. BL21 strains with the merT/merP transporters showed increased sensitivity to mercury concentrations as expected. However, there was no inhibition of growth with wild type BL21 or strains with transporters for lead and nickel even at very high metal concentrations.
  1. Considering cell density, both lead and nickel sequestering strains removed significantly more metal compared with the wild type BL21 strain. The mercury sequestering strain did not sequester significantly more metal compared with the wild type strain.

References


  1. Wilson, D. B. Construction and characterization of Escherichia coli genetically engineered for Construction and Characterization of Escherichia coli Genetically Engineered for Bioremediation of Hg 2 ϩ -Contaminated Environments. 2–6 (1997).
  2. Krishnaswamy, R. & Wilson, D. B. Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II) bioaccumulation. Appl. Environ. Microbiol. 66, 5383–6 (2000).
  3. Huang, J. et al. Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis. Plant Physiol. 158, 1779–88 (2012).