Team:Cornell/project/background/lead

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<h1>Idea</h1>
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Mercury, nickel, and lead were targeted for sequestration by our strain of bacteria by utilizing the efficient binding properties of yeast metallothionein (CRS5) and specificity of the respective metal transport proteins, <i>merT</i> and <i>merP</i><sup>1</sup>, <i>nixA</i><sup>2</sup>, and <i>CBP4</i>. The yeast metallothionein<sup>3</sup> and two heavy metal transport proteins were BioBricked. Our wetlab sub team co-transformed the parts using different plasmids and multiple selection markers to develop functional composite constructs. The idea of utilizing metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this is a novel idea for lead.
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To test the sequestration efficiency of each metal, <i>E.coli</i> BL21-A1 was transformed with the yeast 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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<h1 style="margin-top: 0px;">Health Risks</h1>
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<h1>Components</h1>
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Lead has no known function, and therefore no place, in the human body<sup>[4]</sup>. The lack of any robust, evolved system to deal with lead means that when it enters the organism, it will not be filtered naturally, and instead act as a disruptive, persistent, and often unnoticed antagonist to normal function. What makes lead so insidious? As it accumulates, lead will begin to take the place of other metals in biochemical reactions, replacing zinc or calcium when it is available for chemical reactions. In fact, “Lead binds to calcium-activated proteins with much higher (105 times) affinity than calcium.<sup>[10]</sup>” As a result, 75-90% of lead body load is in mineralizing tissues such as teeth and bones.
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Because of these issues, the United States’ Environmental Protection Agency, which was tasked to set safe levels of chemicals in drinking water by the 1974 Safe Drinking Water Act, has set 0 as the Maximum Contaminant Level Goal for lead. The U.S. Environmental Protection Agency sets the maximum allowable lead concentration at .015 mg/L (74.8 nM)<sup>[6]</sup>. Any concentration above the set maximum requires additional treatment for removal of lead.  On January 4th, 2014 a new provision of the Safe Drinking Water Act requires that any pipe used for the transport of potable water must contain less than 0.25% lead--a reduction from 8% under the previous law.  Lowering levels of lead in piping will help to reduce lead in drinking water - especially since lead piping is the greatest cause of consumed lead in the US - but environmental routes of pollution still exist.
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Lead is especially dangerous for children, as their porous GI tracts, and the increased vulnerability and volatility of their developing body systems make them highly susceptible to the disruptive effects of even small amounts of lead. It also takes them much more time to clear it: the half-life of lead in the adult human body is 1 month, but 10 months in a child’s <sup>[5]</sup>. Low-level exposure can be quite harmful: an increase in blood lead level from 10μg/dL to 20μg/dL is associated with an almost 3-point drop in IQ all on its own<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.
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Common effects in children: Impaired neurological development, gastrointestinal distress, anemia, kidney failure, irritability, lethargy, learning disabilities, erratic behavior.<br>
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Common effects in adults: gastrointestinal distress, weakness, pins and needle, kidney failure <br>
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Extreme cases: neurological damage, death
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<h1>Case Studies</h1>
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<h1>Experiments</h1>
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The growth rates of the sequestering strain were measured using spectrophotometry. In addition, two methods were used to determine heavy metal sequestration efficiency. <br><br>
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<li> Spectrophotometer was used to analyze and compare the kinetic growth rates of E. coli cultures expressing only the metallothionein protein, only the transporter proteins, both metallothionein and transporter proteins, and just the vector backbone as a control. </li>
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<li>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.</li>
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<li>Sequestration efficiency was measured by growing both the wild type strains as well as sequestering strains in different concentrations of heavy metals. The concentration of heavy metals after growth was measured in two ways: </li>
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<li> Nutrient Analysis Lab at Cornell University</li>
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<li> Using green-fluorescent heavy metal indicator Phen Green</li>
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According to the Blacksmith Institute’s 2010 report on the world’s worst pollution problems, lead is the world’s number one toxic threat with an estimated global impact of 18 to 22 million people, more than the population of Syria<sup>[11]</sup>. Lead has long been in use in numerous industries that manufacture products intended for consumption by average families. Famously, tetraethyl lead was added to gasoline (hence leaded gasoline) to improve its octane rating and to increase longevity of motor vehicle components, a practice that began in the United States in 1923, continued through until regulations saw implementation in the 1970s, finally ending with a zero-tolerance ban through the Clear Air Act in 1996<sup>[7]</sup>. A 1988 report to Congress by the Agency for Toxic Substances and Disease Registry estimated that 68 million children had toxic exposure to lead from lead gasoline between 1927-1987.<sup>[7]</sup>
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Other sources of lead include leaded paint, dust that gathers on lead products, contaminated soil, and others. Since lead cannot be absorbed through contact with skin, the metal must be consumed in some form for it to be toxic. Unfortunately, lead tastes sweet. This means that flaking lead paint or the dust that forms on vinyl blinds imported before 1997 might be consumed repeatedly. In fact, the United States Consumer Product Safety Condition found that if a child ingested dust from less than one square inch of blind a day for about 15 to 30 days they could have blood lead levels at or above 10μg/dL <sup>[9]</sup>.
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<h1>Results</h1>
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Lead can usually only enter the body through ingestion, which is why pollution of drinking water supplies is of primary concern.  When ingested at high enough concentrations, lead can be acutely toxic causing neurological damage and death. In 2008, 18 children in Dakar, Senegal died of acute lead poisoning associated with the recycling of lead car batteries.<sup>[2]</sup> Others associated with the recycling facility displayed symptoms ranging from an upset stomach to involuntary convulsions.<sup>[2]</sup>
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<li>BL21 strains with the merT/merP transporters showed increased sensitivity to mercury concentrations as expected. In addition, in high mercury concentrations, wild type strain growth was inhibited more than sequestration strain growths were inhibited. 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. </li>  
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<h1>Current Remediation Techniques</h1>
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<li>Regarding cell density, both lead and nickel sequestering strains removed significantly more metal compared to the wild type BL21 strain. The mercury sequestering strain did not sequester significantly more metal compared to the wild type strain.</li>
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The transport protein being utilized for our project is the calmodulin-binding protein <i>CBP4</i> from <i>Nicotiana tabacum</i>. This protein is structurally similar to non-selective membrane channel proteins from other eukaryotes and has been shown to confer nickel tolerance and lead hypersensitivity.<sup>[1]</sup> Transgenic plants overexpressing <i>NtCBP4</i> were found to have increased uptake of Pb<sup>2+</sup> ions into cells, likely leading to the increased toxicity.<sup>[1]</sup>  While it has been suggested that <i>NtCBP4</i> could possibly be used for bioremediation purposes and other attempts have been made at lead removal from water using genetically engineered organisms, to the best of our knowledge no attempt has been made at utilizing <i>NtCBP4</i> for precisely this purpose.<sup>[1],[2],[3]</sup>.  We believe that the specificity of this transport protein for lead and its readily available sequence make it an ideal candidate for bioremediation.
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<li> Arazi, T., Sunkar, R., Kaplan, B., & Fromm, H. (1999). A tobacco plasma membrane calmodulin-binding transporter confers Ni2 tolerance and Pb2 hypersensitivity in transgenic plants. <i>The Plant Journal</i>, 171-182</li>
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<li>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).</li>
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<li>Song, W., Sohn, E., Martinoia, E., Lee, Y., Yang, Y., Jasinski, M., Forestier, C., Hwang, I., & Lee, Y. (2003). Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nature Biotechnology, 914-919.</li>
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<li>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).</li>
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<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li>
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<li>Huang, J. et al. Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis. Plant Physiol. 158, 1779–88 (2012).</li>
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<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li>
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<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li>
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<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li>
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<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li>
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<li>Schwartz, Joel. "Low-level lead exposure and children′ s IQ: a metaanalysis and search for a threshold." Environmental research 65.1 (1994): 42-55.</li>
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<li>"CPSC Finds Lead Poisoning Hazard for Young Children in Imported Vinyl Miniblinds." U.S. Consumer Product Safety Commission. US Consumer Product Safety Commission, n.d. Web. 15 Oct. 2014.</li>
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<li> "Lead Induced Encephalopathy: An Overview." International Journal of Pharma and Bio Sciences 2.1 (2011): 70-86. Web. http://ijpbs.net/volume2/issue1/pharma/_6.pdf.</li>
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<li> McCartor, A., & Becker, D. (2010). Blacksmith Institute's World's Worst Pollution Problems 2010. Retrieved from: http://www.worstpolluted.org/files/FileUpload/files/2010/WWPP-2010-Report-Web.pdf </li>
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Revision as of 06:37, 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 yeast metallothionein (CRS5) and specificity of the respective metal transport proteins, merT and merP1, nixA2, and CBP4. The yeast metallothionein3 and two heavy metal transport proteins were BioBricked. Our wetlab sub team co-transformed the parts using different plasmids and multiple selection markers to develop functional composite constructs. The idea of utilizing metallothioneins in parallel with metal transporters for sequestration has been studied in depth for mercury and nickel. However, this is a novel idea for lead.

To test the sequestration efficiency of each metal, E.coli BL21-A1 was transformed with the yeast 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.

Components

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. Spectrophotometer was used to analyze and compare the kinetic growth rates of E. coli cultures expressing only the metallothionein protein, only the transporter proteins, both metallothionein and transporter proteins, 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 different concentrations of heavy metals. 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. In addition, in high mercury concentrations, wild type strain growth was inhibited more than sequestration strain growths were inhibited. 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. Regarding cell density, both lead and nickel sequestering strains removed significantly more metal compared to the wild type BL21 strain. The mercury sequestering strain did not sequester significantly more metal compared to 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).

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