Team:Cornell/project/background/lead

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<h1>Idea</h1>
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<h1 style="margin-top: 0px;">Health Risks</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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Lead has no known biological function, and therefore no place in the human body.<sup>[1]</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>[2]</sup> As a result, 75-90% of lead body load is in mineralizing tissues such as teeth and bones.
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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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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>[3]</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 purge it from the body: the half-life of lead in the adult human body is 1 month, but 10 months in a child’s.<sup>[4]</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.<sup>[5]</sup> Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning.<sup>[5]</sup>
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<h1>Components</h1>
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<b><font size=3>Side Effects of Lead Poisoning:</font></b>
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<b>For infants and children:</b>
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<li>Impaired neurological development</li>
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<li>Gastrointestinal distress</li>
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<li>Anemia</li>
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<li>Kidney failure</li>
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<li>Irritability</li>
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<li>Lethargy</li>
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<li>Learning disabilities</li>
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<li>Erratic behavior.</li>
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<b>For adults:</b>
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<li>Gastrointestinal distress</li>
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<li>Weakness</li>
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<li>Pins and needles</li>
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<li>Kidney failure</li>
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<b>Extreme cases of high lead poisoning</b>
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<li>Neurological damage</li>
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<li>Death</li>
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<h1>Experiments</h1>
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<h1>Case Studies</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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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>[6]</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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<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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Other sources of lead include leaded paint, dust that gathers on lead products, and contaminated soil. 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>[8]</sup>  
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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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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>[9]</sup>  Others associated with the recycling facility displayed symptoms ranging from an upset stomach to involuntary convulsions.<sup>[9]</sup>
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<h4>Ithaca Gun Factory:</h4>  
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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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Originally founded in 1883 by Henry Baker, a towering brick smokestack stands as the only remnant of a once-bustling production facility. The Ithaca Gun Company was famous across the world for its shotguns used by Annie Oakley and John Philip Sousa. Throughout its history of production, the factory emitted immense amounts of lead into the surrounding ground. In fact, a 2003 EPA assessment found the need for the removal of 2370 tons of the heavy metal. This mass is roughly equivalent to that of a space shuttle before launch.<sup>[6]</sup> As a result, the Ithaca Gun Company area underwent a lead cleanup project in 2004. However, two years later, surface levels of lead were tested and contamination was as high as 184,000 ppm--460 times the goal set by the EPA for the 2004 cleanup. 
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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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The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since.<sup>[10]</sup> Even more disturbing, this lead contamination is currently located directly next to Ithaca Falls, a popular swimming and fishing site for locals and Cornell students alike. Cornell University, which use to own this property, sold it to the town from $1, so that the EPA could declare it a national Superfunds site, and pay for the cleanup necessary in the years to come.
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<h1>Results</h1>
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<h1>Current Remediation Techniques</h1>
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<b>Water Filters:</b>
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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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The main method currently employed in limiting our consumption of lead via drinking water is through the installation of reverse osmosis, distillation, or chemical (carbon, activated alumina) water filters,<sup>[11]</sup> at scales ranging from industrial to in-home implementation. In homes with lead components in water piping systems, if water has been relatively stagnant for up to 6 hours, it should be flushed through the system to avoid ingesting built-up lead.<sup>[12]</sup> Increased water corrosivity, influenced by pH,<sup>[13]</sup> generally results in a higher lead content.
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<b>Redox media, non-chemical water treatment:</b>
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This fluid treatment passes the lead through a proprietary filter, causing a redox reaction in which “soluble lead cations are reduced to insoluble lead atoms, which are electroplated onto the surface of the media.”<sup>[14]</sup>  
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<b>Guar Gum:</b> Adsorption by this unique compound,<sup>[15]</sup> produced from the ground seeds of guar beans, was sufficient to remove 56.7% of lead from water at a gum concentration of 1,000 parts per million.
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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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<h1><i>cbp4</i></h1>
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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>[10]</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>[10]</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>[9],[10],[16]</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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Lead Data
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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>"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>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>"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>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>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li>
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</ol>
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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> 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> 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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<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>"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> 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> 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> Center for Disease Control. (n.d.). Lead and Drinking Water from Private Wells. Retrieved from http://www.cdc.gov/healthywater/drinking/private/wells/disease/lead.html</li>
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<li> United State Environmental Protection Agency. (1993, June). Actions You Can Take To Reduce Lead In Drinking Water. Retrieved from http://water.epa.gov/drink/info/lead/lead1.cfm </li>
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<li> Penn State Extension. (2014). Lead in Drinking Water. Retrieved from http://extension.psu.edu/natural-resources/water/drinking-water/water-testing/pollutants/lead-in-drinking-water </li>
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<li> KDF Fluid Treatment, Inc. (2014). Removing Lead from Water and Heavy Metal Removal from Water. Retrieved from http://www.kdfft.com/success_metal.htm</li>
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<li> Pal, A. et al. Polyelectrolytic aqueous guar gum for adsorptive separation of soluble Pb(II) from contaminated water. Carbohydr. Polymer. 110, 224–230 (2014). </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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Latest revision as of 03:33, 18 October 2014

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Project Background

Health Risks

Lead has no known biological function, and therefore no place in the human body.[1] 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.”[2] As a result, 75-90% of lead body load is in mineralizing tissues such as teeth and bones.

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).[3] 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.

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 purge it from the body: the half-life of lead in the adult human body is 1 month, but 10 months in a child’s.[4] 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.[5] Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning.[5]
Side Effects of Lead Poisoning:

For infants and children:
  • Impaired neurological development
  • Gastrointestinal distress
  • Anemia
  • Kidney failure
  • Irritability
  • Lethargy
  • Learning disabilities
  • Erratic behavior.
For adults:
  • Gastrointestinal distress
  • Weakness
  • Pins and needles
  • Kidney failure
Extreme cases of high lead poisoning
  • Neurological damage
  • Death

Case Studies

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.[6] 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.[7] 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.[7]

Other sources of lead include leaded paint, dust that gathers on lead products, and contaminated soil. 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.[8]

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.[9] Others associated with the recycling facility displayed symptoms ranging from an upset stomach to involuntary convulsions.[9]

Ithaca Gun Factory:


Originally founded in 1883 by Henry Baker, a towering brick smokestack stands as the only remnant of a once-bustling production facility. The Ithaca Gun Company was famous across the world for its shotguns used by Annie Oakley and John Philip Sousa. Throughout its history of production, the factory emitted immense amounts of lead into the surrounding ground. In fact, a 2003 EPA assessment found the need for the removal of 2370 tons of the heavy metal. This mass is roughly equivalent to that of a space shuttle before launch.[6] As a result, the Ithaca Gun Company area underwent a lead cleanup project in 2004. However, two years later, surface levels of lead were tested and contamination was as high as 184,000 ppm--460 times the goal set by the EPA for the 2004 cleanup. The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since.[10] Even more disturbing, this lead contamination is currently located directly next to Ithaca Falls, a popular swimming and fishing site for locals and Cornell students alike. Cornell University, which use to own this property, sold it to the town from $1, so that the EPA could declare it a national Superfunds site, and pay for the cleanup necessary in the years to come.

Current Remediation Techniques

Water Filters: The main method currently employed in limiting our consumption of lead via drinking water is through the installation of reverse osmosis, distillation, or chemical (carbon, activated alumina) water filters,[11] at scales ranging from industrial to in-home implementation. In homes with lead components in water piping systems, if water has been relatively stagnant for up to 6 hours, it should be flushed through the system to avoid ingesting built-up lead.[12] Increased water corrosivity, influenced by pH,[13] generally results in a higher lead content.
Redox media, non-chemical water treatment: This fluid treatment passes the lead through a proprietary filter, causing a redox reaction in which “soluble lead cations are reduced to insoluble lead atoms, which are electroplated onto the surface of the media.”[14]
Guar Gum: Adsorption by this unique compound,[15] produced from the ground seeds of guar beans, was sufficient to remove 56.7% of lead from water at a gum concentration of 1,000 parts per million.

cbp4

The transport protein being utilized for our project is the calmodulin-binding protein CBP4 from Nicotiana tabacum. 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.[10] Transgenic plants overexpressing NtCBP4 were found to have increased uptake of Pb2+ ions into cells, likely leading to the increased toxicity.[10] While it has been suggested that NtCBP4 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 NtCBP4 for precisely this purpose.[9],[10],[16] We believe that the specificity of this transport protein for lead and its readily available sequence make it an ideal candidate for bioremediation.

References

  1. "Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.
  2. "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
  3. "Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.
  4. "Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.
  5. Schwartz, Joel. "Low-level lead exposure and children′ s IQ: a metaanalysis and search for a threshold." Environmental research 65.1 (1994): 42-55.
  6. 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
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