http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=E.Holmes&year=&month=2014.igem.org - User contributions [en]2024-03-28T22:01:40ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Cornell/project/background/nickelTeam:Cornell/project/background/nickel2014-10-18T03:19:40Z<p>E.Holmes: </p>
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<h1>Health Risks</h1><br />
Nickel is a natural element that constitutes approximately 0.009% of the earth's crust. Nickel sulfides, silicates and oxides are commonly used in mining and natural resources.<sup>[1]</sup> The most common nickel sulfide mineral is pentlandite (NiFe)<sub>9</sub>S<sub>8</sub> accounts for the majority of nickel produced globally.<sup>[2,3]</sup> Domestic nickel production comes from the smelting of natural nickel ores, refining nickel matte, an impure metallic sulfide product from smelting of sulfides of metal ores, reclamation of nickel metal from nickel based or non-nickel based scrap metal, including salvaged machinery, sheet metal, aircraft and other vehicular parts and discarded consumer goods such as batteries. <br />
<br><br><br />
Nickel compounds are used in construction, mining, smelting, electrical equipment manufacturing, and battery and fuel cell production, among numerous other materials. During construction, there is a high risk for nickel contamination. They can also make their way into the household through ceramics since they often form the bond between enamel and iron. <br />
<br><br><br />
Nickel compounds are so toxic because they are highly resistant to corrosion and oxidation in air and aqueous environments; they are resistant to corrosion by organic acids and exposure to chlorine, fluorine, hydrogen chloride and molten salts.<br />
<br><br><br />
Estimated average daily dietary intake is 0.1-0.3 mg/day.<sup>[4,5]</sup> Less than 0.2 mg/day of which is consumed via food and 5-25 ug/day from water.<sup>[2]</sup> Dermal exposure is one of the most common routes of exposure and even low levels of exposure may cause nickel allergic dermatitis.<sup>[6-8]</sup><br />
<br><br><br />
<b>Common Effects</b>:<sup>[1]</sup><br />
<ul><br />
<li>Gastrointestinal distress like: nausea, vomiting, and diarrhea</li><br />
<li>Dermatitis (eczema like effects: rash, itchiness)</li><br />
<li>Neurological effects</li><br />
<li>Nickel specific asthma</li><br />
</ul><br />
<br />
<b>Extreme Cases:</b><br />
<ul><br />
<li> Coma </li><br />
<li> Death </li><br />
</ul><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Case Study</h1><br />
<b>New South Wales, Australia:</b> In 2004, New South Wales, Australia observed a huge spike in nickel concentration in their drinking water. (See graph) Although scientists don't know the exact reasons for how nickel concentrations increased so dramatically, as shown in figure 1, they hypothesize that it could be the result of a natural reduction of flow rate during a period of drought and the subsequent introduction of mine water into the drinking water supply. Overall fluctuations of nickel concentrations over the three years were attributed to natural dilution and changes in demands of water.<br />
<br><br><br />
The Australian Drinking Water Guidelines mandates a safety threshold of 0.02 mg Ni/L water, a value that is based on 70 kg (154 lbs) average body weight, 2 L water consumed daily and 1000 as the safety factor to account for uncertainty of extending animal study results to humans. The residents of New South Wales are assumed to have a similar diet to the rest of Australia's population so that the results of the study can be extended to the whole country. The study also assumed that the entire population of New South Wales was nickel-sensitive. This would lead to a lower Lowest Observed Adverse Effect Level (LOAEL) and set stricter limit for tolerable mean nickel concentrations. The result of the study showed that the mean nickel concentration, 0.03 mg/L with a 95% confidence interval of 0.02-0.04 mg/L, is only approximately 7% of the LOAEL. Thus the mean nickel concentration in drinking water in New South Wales appears to have no health risks.<br />
</div><br />
</div><br />
<div class="col-md-4 col-xs-16"><br />
<div class="thumbnail" style="margin-top: 80px"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Cornell_NickelCaseStudy.jpg"><br />
<div class="caption center"><br />
New South Wales, Austrailia<br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
Although no real risks were detected, the town implemented increased surveillance of nickel concentrations and made plans to use alternative sources to supplement drinking water supplies during droughts. This study shows the importance of continued vigilance in maintaining high water quality standards at all times, had the concentration of nickel increased past the LOAEL, health effects could have been more drastic.<sup>[9]</sup><br />
<br><br />
<div class="center"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c5/NSW_Chart_-_Nickel_Background.png" width="100%"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Cyclic electrowinning/precipitation (CEP) :</b> use of electrical current to transform positively charged metal cations into a stable, solid state where they can be easily separated from water and removed. <br>Drawback: concentration of cations must be high (threshold of 100 ppm)<br />
<br><br><br />
<b>Chemical precipitation:</b> use of hydroxides and sulfides to precipitate cations.<br> Advantages:<ol><li>Well-established, many available chemicals and equipment</li><li>Convenient, self-operating and low-maintenance due to closed system nature</li></ol>Disadvantages:<ol><li>Formation of toxic sludge from precipitate, which is environmentally and economically costly to remove</li><li>Requires extra flocculation/coagulation due to precipitation</li><li>Each metal has a distinct pH for optimum precipitation</li><li>Corrosive chemicals increases safety concerns</li></ol><br />
<b>Ion exchange:</b> reversible chemical reaction where ions from water or wastewater solution are exchanged for similarly charged ions attached to a stationary solid particle that are usually inorganic zeolites or resins.<br />
<br><br><br />
<b>Reverse osmosis:</b> effective molecular filter to remove dissolved solutes through a membrane <br>Advantages:<ol><li>Reduces concentration of all ionic contaminants, not just the heavy metal in question</li><li>Can be scaled up easily</li></ol>Disadvantages:<ol><li>Expensive</li><li>Requires high pressure</li><li>Too sensitive to operating conditions</li></ol><br />
<b>Phytoremediation:</b> use of plants to remediate heavy metals in contaminated soil, sludge, water etc.<br />
<br><br><br />
<b>Microbial remediation:</b> use of microorganisms to degrade hazardous contaminants<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="nixA"><br />
<h1><i>nixA</i></h1><br />
The transport protein being utilized for this project is <i>nixA</i> from <i>Helicobacter pylori</i>. This protein resembles many eukaryotic integral membrane proteins and represents a high-affinity nickel transport system when expressed in <i>E. coli</i>.<sup>[10]</sup> The <i>nixA</i> gene has been introduced into <i>E. coli</i> previously to sequester Ni<sup>2+</sup> from water at 4 times the level of wild type cells.<sup>[11]</sup> We hope to improve upon this system by combining the <i>nixA</i> gene with a different metallothionein than previously used, utilizing a different regulatory system, and creating modular genetic parts. <br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Sullivan, R. J. (Litton Systems, Inc.) Air Pollution Aspects of Nickel and Its Compounds. NTIS No. PB188070. September 1969. p.18.</li><br />
<li>Kirk-Othmer Encyclopedia of Chemical Technology. Third Edition. Volume 15. John Wiley and Sons, Inc. New York. 1980. pp.787-797.</li><br />
<li>Nriagu, J. O. ed. Nickel in the Environment. John Wiley and Sons, Inc., New York. 1980. p. 55.</li><br />
<li>Christensen OB, Lagesson V. Nickel concentration of blood and urine after oral administration. Ann Clin Lab Sci 1981; 11: 119–25.</li><br />
<li>Committee on Toxicity of Chemicals in Food Consumer Products and the Environment. Nickel leaching from kettle elements into boiled water. London: Committee onToxicity; 2003. Available from: http://www.food.gov.uk/multimedia/pdfs/2003-02.pdf (Cited 24 October 2008.)</li><br />
<li>Beattie PE, Green C, Lowe G, Lewis-Jones MS. Which children should we patch test? Clin Exp Dermatol 2006; 32: 6–11.</li><br />
<li>Militello G, Jacob SE, Crawford GH. Allergic contact dermatitis in children. Curr Opin Pediatr 2006; 18: 385–90. doi:10.1097/01.mop.0000236387.56709.6d</li><br />
<li>Silverberg NB, Licht J, Friedler S et al. Nickel contact hypersensitivity in children. Pediatr Dermatol 2002; 19: 110–3. doi:10.1046/j.1525-1470.2002.00057.x</li><br />
<li>Alam, Noore, Stephen J. Corbett, and Helen C. Ptolemy. "Environmental Health Risk Assessment of Nickel Contamination of Drinking Water in a County Town in NSW." <i>NSW Public Health Bulletin</i> (2008): n. pag. Web. http://www.publish.csiro.au/?act=view_file&file_id=NB97043.pdf</li><br />
<li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene nixA: Synthesis of catalytically active urease in <i>Escherichia coli</i> independent of growth conditions. <i>Molecular Microbiology</i>, 97-109.<br />
</li><br />
<li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an <i>Escherichia coli</i> Strain Genetically Engineered for Ni(II) Bioaccumulation. <i>Applied and Environmental Microbiology</i>, 5383-5386.<br />
</li><br />
<br />
</ol><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/nickelTeam:Cornell/project/background/nickel2014-10-18T03:18:46Z<p>E.Holmes: </p>
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<h1>Health Risks</h1><br />
Nickel is a natural element that constitutes approximately 0.009% of the earth's crust. Nickel sulfides, silicates and oxides are commonly used in mining and natural resources.<sup>[1]</sup> The most common nickel sulfide mineral is pentlandite (NiFe)<sub>9</sub>S<sub>8</sub> accounts for the majority of nickel produced globally.<sup>[2,3]</sup> Domestic nickel production comes from the smelting of natural nickel ores, refining nickel matte, an impure metallic sulfide product from smelting of sulfides of metal ores, reclamation of nickel metal from nickel based or non-nickel based scrap metal, including salvaged machinery, sheet metal, aircraft and other vehicular parts and discarded consumer goods such as batteries. <br />
<br><br><br />
Nickel compounds are used in construction, mining, smelting, electrical equipment manufacturing, and battery and fuel cell production, among numerous other materials. During construction, there is a high risk for nickel contamination. They can also make their way into the household through ceramics since they often form the bond between enamel and iron. <br />
<br><br><br />
Nickel compounds are so toxic because they are highly resistant to corrosion and oxidation in air and aqueous environments; they are resistant to corrosion by organic acids and exposure to chlorine, fluorine, hydrogen chloride and molten salts.<br />
<br><br><br />
Estimated average daily dietary intake is 0.1-0.3 mg/day.<sup>[4,5]</sup> Less than 0.2 mg/day of which is consumed via food and 5-25 ug/day from water.<sup>[2]</sup> Dermal exposure is one of the most common routes of exposure and even low levels of exposure may cause nickel allergic dermatitis.<sup>[6-8]</sup><br />
<br><br><br />
<b>Common Effects</b>:<sup>[1]</sup><br />
<ul><br />
<li>Gastrointestinal distress like: nausea, vomiting, and diarrhea</li><br />
<li>Dermatitis (eczema like effects: rash, itchiness)</li><br />
<li>Neurological effects</li><br />
<li>Nickel specific asthma</li><br />
</ul><br />
<br />
<b>Extreme Cases:</b><br />
<ul><br />
<li> Coma </li><br />
<li> Death </li><br />
</ul><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Case Study</h1><br />
<b>New South Wales, Australia:</b> In 2004, New South Wales, Australia observed a huge spike in nickel concentration in their drinking water. (See graph) Although scientists don't know the exact reasons for how nickel concentrations increased so dramatically, as shown in figure 1, they hypothesize that it could be the result of a natural reduction of flow rate during a period of drought and the subsequent introduction of mine water into the drinking water supply. Overall fluctuations of nickel concentrations over the three years were attributed to natural dilution and changes in demands of water.<br />
<br><br><br />
The Australian Drinking Water Guidelines mandates a safety threshold of 0.02 mg Ni/L water, a value that is based on 70 kg (154 lbs) average body weight, 2 L water consumed daily and 1000 as the safety factor to account for uncertainty of extending animal study results to humans. The residents of New South Wales are assumed to have a similar diet to the rest of Australia's population so that the results of the study can be extended to the whole country. The study also assumed that the entire population of New South Wales was nickel-sensitive. This would lead to a lower Lowest Observed Adverse Effect Level (LOAEL) and set stricter limit for tolerable mean nickel concentrations. The result of the study showed that the mean nickel concentration, 0.03 mg/L with a 95% confidence interval of 0.02-0.04 mg/L, is only approximately 7% of the LOAEL. Thus the mean nickel concentration in drinking water in New South Wales appears to have no health risks.<br />
<br><br />
<div class="center"><br />
<img src="https://static.igem.org/mediawiki/2014/c/c5/NSW_Chart_-_Nickel_Background.png" width="100%"><br />
</div><br />
</div><br />
<div class="col-md-4 col-xs-16"><br />
<div class="thumbnail" style="margin-top: 80px"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Cornell_NickelCaseStudy.jpg"><br />
<div class="caption center"><br />
New South Wales, Austrailia<br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
Although no real risks were detected, the town implemented increased surveillance of nickel concentrations and made plans to use alternative sources to supplement drinking water supplies during droughts. This study shows the importance of continued vigilance in maintaining high water quality standards at all times, had the concentration of nickel increased past the LOAEL, health effects could have been more drastic.<sup>[9]</sup><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Cyclic electrowinning/precipitation (CEP) :</b> use of electrical current to transform positively charged metal cations into a stable, solid state where they can be easily separated from water and removed. <br>Drawback: concentration of cations must be high (threshold of 100 ppm)<br />
<br><br><br />
<b>Chemical precipitation:</b> use of hydroxides and sulfides to precipitate cations.<br> Advantages:<ol><li>Well-established, many available chemicals and equipment</li><li>Convenient, self-operating and low-maintenance due to closed system nature</li></ol>Disadvantages:<ol><li>Formation of toxic sludge from precipitate, which is environmentally and economically costly to remove</li><li>Requires extra flocculation/coagulation due to precipitation</li><li>Each metal has a distinct pH for optimum precipitation</li><li>Corrosive chemicals increases safety concerns</li></ol><br />
<b>Ion exchange:</b> reversible chemical reaction where ions from water or wastewater solution are exchanged for similarly charged ions attached to a stationary solid particle that are usually inorganic zeolites or resins.<br />
<br><br><br />
<b>Reverse osmosis:</b> effective molecular filter to remove dissolved solutes through a membrane <br>Advantages:<ol><li>Reduces concentration of all ionic contaminants, not just the heavy metal in question</li><li>Can be scaled up easily</li></ol>Disadvantages:<ol><li>Expensive</li><li>Requires high pressure</li><li>Too sensitive to operating conditions</li></ol><br />
<b>Phytoremediation:</b> use of plants to remediate heavy metals in contaminated soil, sludge, water etc.<br />
<br><br><br />
<b>Microbial remediation:</b> use of microorganisms to degrade hazardous contaminants<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="nixA"><br />
<h1><i>nixA</i></h1><br />
The transport protein being utilized for this project is <i>nixA</i> from <i>Helicobacter pylori</i>. This protein resembles many eukaryotic integral membrane proteins and represents a high-affinity nickel transport system when expressed in <i>E. coli</i>.<sup>[10]</sup> The <i>nixA</i> gene has been introduced into <i>E. coli</i> previously to sequester Ni<sup>2+</sup> from water at 4 times the level of wild type cells.<sup>[11]</sup> We hope to improve upon this system by combining the <i>nixA</i> gene with a different metallothionein than previously used, utilizing a different regulatory system, and creating modular genetic parts. <br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Sullivan, R. J. (Litton Systems, Inc.) Air Pollution Aspects of Nickel and Its Compounds. NTIS No. PB188070. September 1969. p.18.</li><br />
<li>Kirk-Othmer Encyclopedia of Chemical Technology. Third Edition. Volume 15. John Wiley and Sons, Inc. New York. 1980. pp.787-797.</li><br />
<li>Nriagu, J. O. ed. Nickel in the Environment. John Wiley and Sons, Inc., New York. 1980. p. 55.</li><br />
<li>Christensen OB, Lagesson V. Nickel concentration of blood and urine after oral administration. Ann Clin Lab Sci 1981; 11: 119–25.</li><br />
<li>Committee on Toxicity of Chemicals in Food Consumer Products and the Environment. Nickel leaching from kettle elements into boiled water. London: Committee onToxicity; 2003. Available from: http://www.food.gov.uk/multimedia/pdfs/2003-02.pdf (Cited 24 October 2008.)</li><br />
<li>Beattie PE, Green C, Lowe G, Lewis-Jones MS. Which children should we patch test? Clin Exp Dermatol 2006; 32: 6–11.</li><br />
<li>Militello G, Jacob SE, Crawford GH. Allergic contact dermatitis in children. Curr Opin Pediatr 2006; 18: 385–90. doi:10.1097/01.mop.0000236387.56709.6d</li><br />
<li>Silverberg NB, Licht J, Friedler S et al. Nickel contact hypersensitivity in children. Pediatr Dermatol 2002; 19: 110–3. doi:10.1046/j.1525-1470.2002.00057.x</li><br />
<li>Alam, Noore, Stephen J. Corbett, and Helen C. Ptolemy. "Environmental Health Risk Assessment of Nickel Contamination of Drinking Water in a County Town in NSW." <i>NSW Public Health Bulletin</i> (2008): n. pag. Web. http://www.publish.csiro.au/?act=view_file&file_id=NB97043.pdf</li><br />
<li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene nixA: Synthesis of catalytically active urease in <i>Escherichia coli</i> independent of growth conditions. <i>Molecular Microbiology</i>, 97-109.<br />
</li><br />
<li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an <i>Escherichia coli</i> Strain Genetically Engineered for Ni(II) Bioaccumulation. <i>Applied and Environmental Microbiology</i>, 5383-5386.<br />
</li><br />
<br />
</ol><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/nickelTeam:Cornell/project/background/nickel2014-10-18T03:17:51Z<p>E.Holmes: </p>
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<div class="col-md-12 col-xs-18"><br />
<h1>Health Risks</h1><br />
Nickel is a natural element that constitutes approximately 0.009% of the earth's crust. Nickel sulfides, silicates and oxides are commonly used in mining and natural resources.<sup>[1]</sup> The most common nickel sulfide mineral is pentlandite (NiFe)<sub>9</sub>S<sub>8</sub> accounts for the majority of nickel produced globally.<sup>[2,3]</sup> Domestic nickel production comes from the smelting of natural nickel ores, refining nickel matte, an impure metallic sulfide product from smelting of sulfides of metal ores, reclamation of nickel metal from nickel based or non-nickel based scrap metal, including salvaged machinery, sheet metal, aircraft and other vehicular parts and discarded consumer goods such as batteries. <br />
<br><br><br />
Nickel compounds are used in construction, mining, smelting, electrical equipment manufacturing, and battery and fuel cell production, among numerous other materials. During construction, there is a high risk for nickel contamination. They can also make their way into the household through ceramics since they often form the bond between enamel and iron. <br />
<br><br><br />
Nickel compounds are so toxic because they are highly resistant to corrosion and oxidation in air and aqueous environments; they are resistant to corrosion by organic acids and exposure to chlorine, fluorine, hydrogen chloride and molten salts.<br />
<br><br><br />
Estimated average daily dietary intake is 0.1-0.3 mg/day.<sup>[4,5]</sup> Less than 0.2 mg/day of which is consumed via food and 5-25 ug/day from water.<sup>[2]</sup> Dermal exposure is one of the most common routes of exposure and even low levels of exposure may cause nickel allergic dermatitis.<sup>[6-8]</sup><br />
<br><br><br />
<b>Common Effects</b>:<sup>[1]</sup><br />
<ul><br />
<li>Gastrointestinal distress like: nausea, vomiting, and diarrhea</li><br />
<li>Dermatitis (eczema like effects: rash, itchiness)</li><br />
<li>Neurological effects</li><br />
<li>Nickel specific asthma</li><br />
</ul><br />
<br />
<b>Extreme Cases:</b><br />
<ul><br />
<li> Coma </li><br />
<li> Death </li><br />
</ul><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Case Study</h1><br />
<b>New South Wales, Australia:</b> In 2004, New South Wales, Australia observed a huge spike in nickel concentration in their drinking water. (See graph) Although scientists don't know the exact reasons for how nickel concentrations increased so dramatically, as shown in figure 1, they hypothesize that it could be the result of a natural reduction of flow rate during a period of drought and the subsequent introduction of mine water into the drinking water supply. Overall fluctuations of nickel concentrations over the three years were attributed to natural dilution and changes in demands of water.<br />
<br><br><br />
The Australian Drinking Water Guidelines mandates a safety threshold of 0.02 mg Ni/L water, a value that is based on 70 kg (154 lbs) average body weight, 2 L water consumed daily and 1000 as the safety factor to account for uncertainty of extending animal study results to humans. The residents of New South Wales are assumed to have a similar diet to the rest of Australia's population so that the results of the study can be extended to the whole country. The study also assumed that the entire population of New South Wales was nickel-sensitive. This would lead to a lower Lowest Observed Adverse Effect Level (LOAEL) and set stricter limit for tolerable mean nickel concentrations. The result of the study showed that the mean nickel concentration, 0.03 mg/L with a 95% confidence interval of 0.02-0.04 mg/L, is only approximately 7% of the LOAEL. Thus the mean nickel concentration in drinking water in New South Wales appears to have no health risks.<br />
<br><br />
<img src="https://static.igem.org/mediawiki/2014/c/c5/NSW_Chart_-_Nickel_Background.png" width="100%"><br />
</div><br />
<div class="col-md-4 col-xs-16"><br />
<div class="thumbnail" style="margin-top: 80px"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Cornell_NickelCaseStudy.jpg"><br />
<div class="caption center"><br />
New South Wales, Austrailia<br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
Although no real risks were detected, the town implemented increased surveillance of nickel concentrations and made plans to use alternative sources to supplement drinking water supplies during droughts. This study shows the importance of continued vigilance in maintaining high water quality standards at all times, had the concentration of nickel increased past the LOAEL, health effects could have been more drastic.<sup>[9]</sup><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Cyclic electrowinning/precipitation (CEP) :</b> use of electrical current to transform positively charged metal cations into a stable, solid state where they can be easily separated from water and removed. <br>Drawback: concentration of cations must be high (threshold of 100 ppm)<br />
<br><br><br />
<b>Chemical precipitation:</b> use of hydroxides and sulfides to precipitate cations.<br> Advantages:<ol><li>Well-established, many available chemicals and equipment</li><li>Convenient, self-operating and low-maintenance due to closed system nature</li></ol>Disadvantages:<ol><li>Formation of toxic sludge from precipitate, which is environmentally and economically costly to remove</li><li>Requires extra flocculation/coagulation due to precipitation</li><li>Each metal has a distinct pH for optimum precipitation</li><li>Corrosive chemicals increases safety concerns</li></ol><br />
<b>Ion exchange:</b> reversible chemical reaction where ions from water or wastewater solution are exchanged for similarly charged ions attached to a stationary solid particle that are usually inorganic zeolites or resins.<br />
<br><br><br />
<b>Reverse osmosis:</b> effective molecular filter to remove dissolved solutes through a membrane <br>Advantages:<ol><li>Reduces concentration of all ionic contaminants, not just the heavy metal in question</li><li>Can be scaled up easily</li></ol>Disadvantages:<ol><li>Expensive</li><li>Requires high pressure</li><li>Too sensitive to operating conditions</li></ol><br />
<b>Phytoremediation:</b> use of plants to remediate heavy metals in contaminated soil, sludge, water etc.<br />
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<b>Microbial remediation:</b> use of microorganisms to degrade hazardous contaminants<br />
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<h1><i>nixA</i></h1><br />
The transport protein being utilized for this project is <i>nixA</i> from <i>Helicobacter pylori</i>. This protein resembles many eukaryotic integral membrane proteins and represents a high-affinity nickel transport system when expressed in <i>E. coli</i>.<sup>[10]</sup> The <i>nixA</i> gene has been introduced into <i>E. coli</i> previously to sequester Ni<sup>2+</sup> from water at 4 times the level of wild type cells.<sup>[11]</sup> We hope to improve upon this system by combining the <i>nixA</i> gene with a different metallothionein than previously used, utilizing a different regulatory system, and creating modular genetic parts. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<li>Sullivan, R. J. (Litton Systems, Inc.) Air Pollution Aspects of Nickel and Its Compounds. NTIS No. PB188070. September 1969. p.18.</li><br />
<li>Kirk-Othmer Encyclopedia of Chemical Technology. Third Edition. Volume 15. John Wiley and Sons, Inc. New York. 1980. pp.787-797.</li><br />
<li>Nriagu, J. O. ed. Nickel in the Environment. John Wiley and Sons, Inc., New York. 1980. p. 55.</li><br />
<li>Christensen OB, Lagesson V. Nickel concentration of blood and urine after oral administration. Ann Clin Lab Sci 1981; 11: 119–25.</li><br />
<li>Committee on Toxicity of Chemicals in Food Consumer Products and the Environment. Nickel leaching from kettle elements into boiled water. London: Committee onToxicity; 2003. Available from: http://www.food.gov.uk/multimedia/pdfs/2003-02.pdf (Cited 24 October 2008.)</li><br />
<li>Beattie PE, Green C, Lowe G, Lewis-Jones MS. Which children should we patch test? Clin Exp Dermatol 2006; 32: 6–11.</li><br />
<li>Militello G, Jacob SE, Crawford GH. Allergic contact dermatitis in children. Curr Opin Pediatr 2006; 18: 385–90. doi:10.1097/01.mop.0000236387.56709.6d</li><br />
<li>Silverberg NB, Licht J, Friedler S et al. Nickel contact hypersensitivity in children. Pediatr Dermatol 2002; 19: 110–3. doi:10.1046/j.1525-1470.2002.00057.x</li><br />
<li>Alam, Noore, Stephen J. Corbett, and Helen C. Ptolemy. "Environmental Health Risk Assessment of Nickel Contamination of Drinking Water in a County Town in NSW." <i>NSW Public Health Bulletin</i> (2008): n. pag. Web. http://www.publish.csiro.au/?act=view_file&file_id=NB97043.pdf</li><br />
<li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene nixA: Synthesis of catalytically active urease in <i>Escherichia coli</i> independent of growth conditions. <i>Molecular Microbiology</i>, 97-109.<br />
</li><br />
<li>Krishnaswamy, R., & Wilson, D. (2000). Construction and Characterization of an <i>Escherichia coli</i> Strain Genetically Engineered for Ni(II) Bioaccumulation. <i>Applied and Environmental Microbiology</i>, 5383-5386.<br />
</li><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/metallothioneinTeam:Cornell/project/wetlab/metallothionein2014-10-18T03:05:25Z<p>E.Holmes: </p>
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<h1>Construct Design</h1><br />
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><br />
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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.<br />
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<h1>Results</h1><br />
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 <i>E.coli</i> 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.<br />
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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. <br />
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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. <br />
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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, <b>there is growth</b>. In non-engineered BL21 there is none. This suggests that, in fact, our construct is conferring resistance to metal toxicity to engineered cells.<br />
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<h4>Combination with other BioBricks:</h4><br />
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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 <a href="https://2014.igem.org/Team:Cornell/project/wetlab/nickel">nickel</a>, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/mercury">mercury</a>, and <a href="https://2014.igem.org/Team:Cornell/project/wetlab/lead">lead</a> pages. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<li>Peterson, C., Narula, S., & Armitage, I. (1996). 3D solution structure of copper and silver-substituted yeast metallothioneins. FEBS Letters, 85-93.</li><br />
<li>Davis, Stephanie R., "Characterizing the role of the bacterial metallothionein, SmtA, in mammalian infection" (2011). Honors Scholar Theses. University of Connecticut. Paper 178</li><br />
<li>Chen, S., & Wilson, D. (1997). Construction and characterization of <i>Escherichia coli</i> genetically engineered for bioremediation of Hg(2+)-contaminated environments. <i>Applied and Environmental Microbiology</i>, 63(6), 2442-2445.</li><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/mercuryTeam:Cornell/project/wetlab/mercury2014-10-18T02:56:17Z<p>E.Holmes: </p>
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<h1>Construct Design</h1><br />
To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.<sup>[1]</sup> The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells.<sup>[2]</sup> <br />
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Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-<i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. <br />
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<h3>BBa_K1460007</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). <br />
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What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. <br />
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Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury 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.<br />
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There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and GST-<i>crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.</li><br />
<li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.</li><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/nickelTeam:Cornell/project/wetlab/nickel2014-10-18T02:53:47Z<p>E.Holmes: </p>
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<h1>Construct Design</h1><br />
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><br />
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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. <br />
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<h3>BBa_K1460006</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>nixA</i> should be transporting more nickel ions past the cell wall. This would lead to increased nickel sensitivity. To test for nickel sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460003 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with 1 mM Ni. <br />
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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 <i>nixA</i> 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.<br />
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Part BBa_K1460003 in pUC57 was co-transformed with part <a href="https://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. <br />
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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. <br />
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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="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.<br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<li>Fulkerson, J., & Mobley, H. (2000). Membrane Topology of the <i>nixA</i> Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III. Journal of Bacteriology, 1722-1730.</li><br />
<li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene <i>nixA</i>: Synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Molecular Microbiology, 97-109.</li><br />
<li>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.</li><br />
</ol><br />
</div><br />
</div><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:51:41Z<p>E.Holmes: </p>
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<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filter/pipe system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
An Arduino was used to control the operation of the pump. The Arduino and accompanying circuitry detect the presence of water and turn the pump on. Conversely, they turn the pump off when no water is entering the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
Below is the water-sensing circuit that sends feedback to the Arduino:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at one of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from water, the entire circuit was housed in a Corning pipette box. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/File:Cornell_Nickel2.pngFile:Cornell Nickel2.png2014-10-18T02:49:29Z<p>E.Holmes: uploaded a new version of &quot;File:Cornell Nickel2.png&quot;</p>
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<div></div>E.Holmeshttp://2014.igem.org/File:Cornell_lead2.pngFile:Cornell lead2.png2014-10-18T02:48:25Z<p>E.Holmes: uploaded a new version of &quot;File:Cornell lead2.png&quot;</p>
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<div></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylabTeam:Cornell/project/drylab2014-10-18T02:42:35Z<p>E.Holmes: </p>
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<div class="col-md-8 col-xs-12"><br />
<h1>How it Works</h1><br />
The dry lab component of this year’s project was designed with applicability in mind. Designing for feasibility of scale and taking into account the limitations of the biological components of our filter idea, we settled on a system designed to remove heavy metals from factory waste pipes. This was viewed as one of the most effective potential uses for our water filter system due to the high concentration of pollutants in factory waste and the relatively low volume of water that would need to be filtered. <br />
<br><br><br />
The system is designed to continuously flow contaminated water through our genetically engineered cells while simultaneously preventing their release into the environment.<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3c/800px-CORNELL2014_CAD_%282%29.jpg"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail" style="margin-top: 0px;"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" width="100%"><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-12"> <br />
Contaminated waste water exiting the industrial pipe is directed into a collection bucket. This stored water is pumped into an environmentally robust casing housing our filtration system. Once water enters the system, the water detection circuit turns on the battery via Arduino. The 12 V battery (which can be recharged using an attached solar panel) powers the 800 mA pump, which propels water through the system. The water flows through a carbon water filter to remove any particulates that may clog the more intricate hollow fiber reactor. <br />
<br><br><br />
The hollow fiber reactor is a unit that contains hundreds of small, porous tubes – the hollow fibers – inside an outer casing. In our system, cells are placed in the outer casing and contaminated water flows through the fibers. The pores in the fibers are large enough that metal ions and water can pass through, but cells and larger proteins cannot. As water flows through the cartridge, the ions will naturally diffuse through the fibers where they can come in contact with our modified cells, which then will sequester them as explained in our <a href="https://2014.igem.org/Team:Cornell/project/wetlab">wet lab section</a>.<br />
After water passes through the hollow fiber reactor, it should be clear of most metal contaminants and is free to re-enter the main water stream. When implemented, downstream filters would be incorporated into the system to monitor metal concentrations <a href="https://2014.igem.org/Team:Cornell/project/wetlab/reporters">(using the devised reporter system)</a> and to visually indicate when the metallothionein proteins are saturated.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylabTeam:Cornell/project/drylab2014-10-18T02:40:59Z<p>E.Holmes: </p>
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<div class="container"><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>How it Works</h1><br />
The dry lab component of this year’s project was designed with applicability in mind. Designing for feasibility of scale and taking into account the limitations of the biological components of our filter idea, we settled on a system designed to remove heavy metals from factory waste pipes. This was viewed as one of the most effective potential uses for our water filter system due to the high concentration of pollutants in factory waste and the relatively low volume of water that would need to be filtered. <br />
<br><br><br />
The system is designed to continuously flow contaminated water through our genetically engineered cells while simultaneously preventing their release into the environment.<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3c/800px-CORNELL2014_CAD_%282%29.jpg"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail" style="margin-top: 0px;"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" width="100%"><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-12"> <br />
Contaminated waste water exiting the industrial pipe is directed into a collection bucket. This stored water is pumped into an environmentally robust casing housing our filtration system. Once water enters the system, the water detection circuit turns on the battery via Arduino. The 12 V battery (which can be recharged using an attached solar panel) powers the 800 mA pump, which propels water through the system. The water flows through a carbon water filter to remove any particulates that may clog the more intricate hollow fiber reactor. <br />
<br><br><br />
The hollow fiber reactor is a unit that contains hundreds of small, porous tubes – the hollow fibers – inside an outer casing. In our system, cells are placed in the outer casing and contaminated water flows through the fibers. The pores in the fibers are large enough that metal ions and water can pass through, but cells and larger proteins cannot. As water flows through the cartridge, the ions will naturally diffuse through the fibers where they can come in contact with our modified cells, which then will sequester them as explained in our <a href="https://2014.igem.org/Team:Cornell/project/wetlab">wet lab section</a>.<br />
After water passes through the hollow fiber reactor, it should be clear of most metal contaminants and is free to re-enter the main water stream. When implemented, downstream filters would be incorporated into the system to monitor metal concentrations <a href="https://2014.igem.org/Team:Cornell/project/wetlab/reporters">(using the devised reporter system)</a> and to ensure there is no release of genetically modified organisms into the environment.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylabTeam:Cornell/project/drylab2014-10-18T02:40:30Z<p>E.Holmes: </p>
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<div>{{:Team:Cornell/header}}<br />
{{:Team:Cornell/project/drylab/header}}<br />
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});<br />
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<body><br />
<div class="container"><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>How it Works</h1><br />
The dry lab component of this year’s project was designed with applicability in mind. Designing for feasibility of scale and taking into account the limitations of the biological components of our filter idea, we settled on a system designed to remove heavy metals from factory waste pipes. This was viewed as one of the most effective potential uses for our water filter system due to the high concentration of pollutants in factory waste and the relatively low volume of water that would need to be filtered. <br />
<br><br><br />
The system is designed to continuously flow contaminated water through our genetically engineered cells while simultaneously preventing their release into the environment.<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3c/800px-CORNELL2014_CAD_%282%29.jpg"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-6"><br />
<div class="thumbnail" style="margin-top: 0px;"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" width="100%"><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-12"> <br />
Contaminated waste water exiting the industrial pipe is directed into a collection bucket. This stored water is pumped into an environmentally robust casing housing our filtration system. Once water enters the system, the water detection circuit turns on the battery via Arduino. The 12 V battery (which can be recharged using an attached solar panel) powers the 800 mA pump, which propels water through the system. The water flows through a carbon water filter to remove any particulates that may clog the more intricate hollow fiber reactor. <br />
<br><br><br />
The hollow fiber reactor is a unit that contains hundreds of small, porous tubes – the hollow fibers – inside an outer casing. In our system, cells are placed in the outer casing and contaminated water flows through the fibers. The pores in the fibers are large enough that metal ions and water can pass through, but cells and larger proteins cannot. As water flows through the cartridge, the ions will naturally diffuse through the fibers where they can come in contact with our modified cells, which then will sequester them as explained in our <a href="https://2014.igem.org/Team:Cornell/project/wetlab">wet lab section</a>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
After water passes through the hollow fiber reactor, it should be clear of most metal contaminants and is free to re-enter the main water stream. When implemented, downstream filters would be incorporated into the system to monitor metal concentrations <a href="https://2014.igem.org/Team:Cornell/project/wetlab/reporters">(using the devised reporter system)</a> and to ensure there is no release of genetically modified organisms into the environment.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:38:50Z<p>E.Holmes: </p>
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<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filter/pipe system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
An Arduino was used to control the operation of the pump. The Arduino and accompanying circuitry detect the presence of water and turn the pump on. Conversely, they turn the pump off when no water is entering the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
Below is the water-sensing circuit that sends feedback to the Arduino:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at one of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:38:08Z<p>E.Holmes: </p>
<hr />
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{{:Team:Cornell/project/drylab/header}}<br />
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<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filter/pipe system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
An Arduino was used to control the operation of the pump. The Arduino and accompanying circuitry detect the presence of water and turn the pump on. Conversely, they turn the pump off when no water is entering the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
Below is the water-sensing circuit that sends feedback to the Arduino:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at 1 of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:37:24Z<p>E.Holmes: </p>
<hr />
<div>{{:Team:Cornell/header}}<br />
{{:Team:Cornell/project/drylab/header}}<br />
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<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.p_dry_elec').addClass('active');<br />
});<br />
</script><br />
<body><br />
<div class="container"><br />
<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filter/pipe system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
An Arduino was used to control the operation of the pump. The Arduino and accompanying circuitry detect the presence of water and turn the pump on. Conversely, they turn the pump off when no water is entering the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
The sensor that was used to give input to the Arduino about if there was water or not is in this circuit below:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at 1 of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:36:48Z<p>E.Holmes: </p>
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<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filter/pipe system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
To control when to pump in water through the filter, an Arduino was used to detect if water was in the system. If there was water in the system, the motor would be pumping water through. If somehow there was no more water left to be pumped, the Arduino would be able to detect this, and shut off the motor until there is some more water in the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
The sensor that was used to give input to the Arduino about if there was water or not is in this circuit below:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at 1 of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:36:09Z<p>E.Holmes: </p>
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<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filter system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump broke. There needs to be a way to control how much water should be running through the filtering/piping system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
To control when to pump in water through the filter, an Arduino was used to detect if water was in the system. If there was water in the system, the motor would be pumping water through. If somehow there was no more water left to be pumped, the Arduino would be able to detect this, and shut off the motor until there is some more water in the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
The sensor that was used to give input to the Arduino about if there was water or not is in this circuit below:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at 1 of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/electronicsTeam:Cornell/project/drylab/electronics2014-10-18T02:34:56Z<p>E.Holmes: </p>
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<div class="row"><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Electronics - Water Level Sensing Circuit</h1><br />
<b>Why do we need it?</b><br><br />
If the entire filtering system was built without the circuit, the water circulation pump would keep on going until either the battery ran out of power or the pump breaks. There needs to be a way to control how much water should be running through the filtering/piping system. That is what the water-sensing circuit is for. <br />
<br><br><br />
<b>General Operation:</b><br />
<br><br />
To control when to pump in water through the filter, an Arduino was used to detect if water was in the system. If there was water in the system, the motor would be pumping water through. If somehow there was no more water left to be pumped, the Arduino would be able to detect this, and shut off the motor until there is some more water in the system. <br />
<br><br><br />
<b>Setup and Operation:</b><br />
<br><br />
The sensor that was used to give input to the Arduino about if there was water or not is in this circuit below:<br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4f/CORNELL2014_Modifiedcircuit.png" style="max-height: 300px;"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3e/CORNELL2014_Circuitdiagram.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
There is an infrared LED (L1) shining on a phototransistor (Q1). Physically, the transparent tubing would be placed in between the infrared LED and phototransistor, and there would be a certain amount of infrared light reaching the phototransistor. In general, a phototransistor produces current proportional to the amount of light shining on it. In this case, the phototransistor that was chosen was more sensitive to the infrared spectrum, so it will produce more current when there is more infrared light reaching it and less current when less infrared light is shining on it. <br />
<br><br><br />
The transparent tubing will either have water running through it or not. When it has some water going through the tube, the water absorbs more of the infrared light between the LED and the phototransistor, so the reading on the voltage should be much lower than when there is no water going through the tubing. <br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/d/d8/CORNELL2014_InsideCircuit.JPG/800px-CORNELL2014_InsideCircuit.JPG"><br />
</div><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/5b/CORNELL2014_EntireCircuit.JPG/800px-CORNELL2014_EntireCircuit.JPG"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
The Arduino Fio is connected at 1 of the outputs of the phototransistor to read the voltage at that point. The Arduino Fio is programmed to cut power to the water circulation pump if there is no water in the system. A voltage threshold is set within the program to determine what voltage read at the phototransistor output is considered to have water or no water in the system. <br />
<br><br><br />
To ensure that the circuit is protected from the water, the entire circuit was housed in Corning pipette box to contain it from water. Holes were drilled to move the infrared LED and phototransistor through the box and to connect the motor with the circuit.<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/functionalreqTeam:Cornell/project/drylab/functionalreq2014-10-18T02:33:37Z<p>E.Holmes: </p>
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{{:Team:Cornell/project/drylab/header}}<br />
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<h1>Functional Requirements</h1><br />
<ol><br />
<li><br />
<b>Filter Out Heavy Metals</b><br />
Our system needed to allow diffusion of heavy metal ions across a filter boundary without letting the genetically engineered cells out into the environment. To satisfy this requirement, we bought a hollow fiber reactor with a molecular weight cut-off (MWCO) of 20 kd @ 50%, effectively isolating the E.coli cells (0.7-1.4 micrometers) from the outlet<sup>[1]</sup>, while allowing diffusion of smaller molecules such as ions across the membrane of the fibers.<br />
</li><br />
<br />
<li> <br />
<b>Isolated System</b><br />
The entire filter system is housed in a watertight box to prevent contaminants from entering. To filter out debris that could enter via the collection bucket, a carbon filter is placed in the system before the fiber reactor. This ensures that only water with microscopic contaminants enters the fiber reactor. Thus, the system is as isolated from the outside environment as possible. <br />
</li><br />
<li><br />
<b>Effective Flow Rates</b> <br />
A carbon water filter was added before the hollow fiber reactor in order to filter out large particles and debris that would clog the hollow fiber reactor and to purify the water of certain compounds which would harm the cells. Entering water needed to have an initial flow rate that would allow it to pass through both filters while still allowing time for heavy metal diffusion across the fiber reactor membrane and uptake by the cells. The pump was placed before the carbon fiber in order to move it effectively through the carbon fiber and still have a slow flow rate through the fiber reactor. <br />
</li><br />
<li> <br />
<b>Saturation Detection</b><br />
A subsystem of reporter constructs was set up inside the box in order to detect when the metallothionein in the fiber reactor is saturated with heavy metals and the cells need replacing. The constructs consisted of a chromoprotein, amilCP, downstream from a specific heavy metal inducible promoter. When the metallothioneins became concentrated, the cells turn visibly blue, indicating that the cells need to be replaced.<br />
</li><br />
<li> <br />
<b>Maintenance</b> <br />
Our system is powered by a high capacity car battery that is recharged by a compact solar panel. Since our flow requirements are not high, the system can last for up to 2 weeks without any sunlight at all. With several hours of sunlight a day, the system can retain charge for months at a time. Combined with the tough, weatherproof design, this allows our system to purify water fully autonomously for very long periods. <br />
</li><br />
</ol> <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>References</h1><br />
<hr><br />
<ol><br />
<li>Nelson DE, Young KD. Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli. J Bacteriol. 2000 Mar182(6):1714-21 p.1719</li><br />
</ol><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/componentsTeam:Cornell/project/drylab/components2014-10-18T02:31:28Z<p>E.Holmes: </p>
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{{:Team:Cornell/project/drylab/header}}<br />
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<div class="col-md-12 col-xs-18"><br />
<h1>List of Components</h1><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg" data-toggle="lightbox" class="thumbnail> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg""><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG"><br />
<div class="caption"><br />
<b>Waterproof Pelican Case (2’x3’)</b><br><br />
The box is a weather-resistant casing for our filter system components. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" style="height: 559px;"><br />
<div class="caption"><br />
<b>Pentek Microguard Filtration System<br></b><br />
Micron Rating: 0.15 Absolute Filtration<br><br />
Pressure Range: 30-125 psi<br><br />
Temperature Range: 40-125 F<br><br />
Flow Rate: 1 GPM<br><br />
Capacity: 200-600 gallons<br><br />
Inlet/Outlet: 1/2"<br><br />
</div><br />
</a><br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG"><br />
<div class="caption"><br />
<b>Collecting Drum (12”x12”)</b><br><br />
The collecting drum is used to collect industrial wastewater from factory outlets. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG"><br />
<div class="caption"><br />
<b>DC Water Circulation Pump<br></b><br />
Voltage: 12 V, Current: 0.8 A <br><br><br />
A DC water circulation pump used to maintain constant flow through the box. The pump is connected to and is controlled by the Arduino.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG"><br />
<div class="caption"><br />
<b>Hollow Fiber Reactor</b><br><br />
Model #: 2011 <br><br />
Fiber Material: high flux polysulfone <br><br />
Pore Size: MWCO 20kd @ 50% <br><br />
20 mL capacity of ECS <br><br><br />
The hollow fiber reactor allows heavy metal ions to flow transversely through a membrane into a compartment with engineered cells, which sequester them from the water. <br />
</div><br />
</a><br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG"><br />
<div class="caption"><br />
<b>Leoch Lead Acid Battery (LPG 12-100)<br></b><br />
Model #: LPG12-100<br><br />
Power Rating: 100 Ah/1200 Whr<br><br />
Voltage: 12 V<br><br />
Dimensions: 13” x 6.8” x 8.4”<br><br><br />
The battery is connected to the Arduino and the pump.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" style="height: 250px;"><br />
<div class="caption"><br />
<b>Instaspark 15 W Mono-Crystalline Solar Panel<br></b><br />
15 W mono-crystalline solar panel<br><br />
Model #: SPCC-15W <br><br><br />
The solar panel is used to recharge the battery.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" style="height: 254px;"><br />
<div class="caption"><br />
<b>Foam and acrylic for keeping components fixed within Pelican Case</b><br><br />
This padding material will hold items like the battery and filters in place and elevate them to an easily accessible area for maintenance. Grooves have been carved in the material to enable for easy monitoring of piping. <br />
</div><br />
</a><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/componentsTeam:Cornell/project/drylab/components2014-10-18T02:29:03Z<p>E.Holmes: </p>
<hr />
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<h1>List of Components</h1><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg" data-toggle="lightbox" class="thumbnail> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg""><br />
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<a href="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG"><br />
<div class="caption"><br />
<b>Waterproof Pelican Case (2’x3’)</b><br><br />
The box is a weather-resistant casing for our filter system components. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" style="height: 559px;"><br />
<div class="caption"><br />
<b>Pentek Microguard Filtration System<br></b><br />
Micron Rating: 0.15 Absolute Filtration<br><br />
Pressure Range: 30-125 psi<br><br />
Temperature Range: 40-125 F<br><br />
Flow Rate: 1 GPM<br><br />
Capacity: 200-600 gallons<br><br />
Inlet/Outlet: 1/2"<br><br />
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<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG"><br />
<div class="caption"><br />
<b>Collecting Drum (12”x12”)</b><br><br />
The collecting drum is used to collect industrial waste from factory outlets. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG"><br />
<div class="caption"><br />
<b>DC Water Circulation Pump<br></b><br />
Voltage: 12 V, Current: 0.8 A <br><br><br />
A DC water circulation pump used to maintain constant flow through the box. The pump is connected to and is controlled by the Arduino.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG"><br />
<div class="caption"><br />
<b>Hollow Fiber Reactor</b><br><br />
Model #: 2011 <br><br />
Fiber Material: high flux polysulfone <br><br />
Pore Size: MWCO 20kd @ 50% <br><br />
20 mL capacity of ECS <br><br><br />
The hollow fiber reactor allows heavy metal ions to flow transversely through a membrane into a compartment with engineered cells, which sequester them from the water. <br />
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<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG"><br />
<div class="caption"><br />
<b>Battery <br></b><br />
Model #: LPG12-100<br><br />
Power Rating: 100 Ah/1200 Whr<br><br />
Voltage: 12 V<br><br />
Dimensions: 13” x 6.8” x 8.4”<br><br><br />
The battery is connected to the Arduino and the pump.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" style="height: 250px;"><br />
<div class="caption"><br />
<b>Solar panel<br></b><br />
15 W mono-crystalline solar panel<br><br />
Model #: SPCC-15W <br><br><br />
The solar panel is used to recharge the battery.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" style="height: 254px;"><br />
<div class="caption"><br />
<b>Foam and Acrylic for keeping components fixed within Pelican Case</b><br><br />
This padding material will hold items like the battery and filters in place and elevate them to an easily accessible area for maintenance. Grooves have been carved in the material to enable for easy monitoring of piping. <br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/drylab/componentsTeam:Cornell/project/drylab/components2014-10-18T02:26:40Z<p>E.Holmes: </p>
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<h1>List of Components</h1><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg" data-toggle="lightbox" class="thumbnail> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/6/64/CORNELL2014_CAD.jpg/800px-CORNELL2014_CAD.jpg""><br />
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<a href="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/8/8d/CORNELL2014_PelicanBox.JPG"><br />
<div class="caption"><br />
<b>Waterproof Pelican Case (2’x3’)</b><br><br />
The box is a weather-resistant casing for our filter system components. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/4/49/CORNELL2014_Filter2.JPG/450px-CORNELL2014_Filter2.JPG" style="height: 559px;"><br />
<div class="caption"><br />
<b>Large Water Filter-.1um<br></b><br />
Micron Rating: 0.15 Absolute Filtration<br><br />
Pressure Range: 30-125 psi<br><br />
Temperature Range: 40-125 F<br><br />
Flow Rate: 1 GPM<br><br />
Capacity: 200-600 gallons<br><br />
Inlet/Outlet: 1/2"<br><br />
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</a><br />
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<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9b/CORNELL2014_Bucket2.JPG"><br />
<div class="caption"><br />
<b>Collecting Drum (12”x12”)</b><br><br />
The collecting drum is used to collect industrial waste from factory outlets. <br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/9/9f/CORNELL2014_Pump.JPG"><br />
<div class="caption"><br />
<b>Pump<br></b><br />
Voltage: 12 V, Current: 0.8 A <br><br><br />
A DC water circulation pump used to maintain constant flow through the box. The pump is connected to and is controlled by the Arduino.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/3/32/CORNELL2014_Fiber1.JPG"><br />
<div class="caption"><br />
<b>Hollow Fiber Reactor</b><br><br />
Model #: 2011 <br><br />
Fiber Material: high flux polysulfone <br><br />
Pore Size: MWCO 20kd @ 50% <br><br />
20 mL capacity of ECS <br><br><br />
The hollow fiber reactor allows heavy metal ions to flow transversely through a membrane into a compartment with engineered cells, which sequester them from the water. <br />
</div><br />
</a><br />
</div><br />
<div class="col-md-4 col-xs-6"><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/f/f3/CORNELL2014_Battery.JPG/800px-CORNELL2014_Battery.JPG"><br />
<div class="caption"><br />
<b>Battery <br></b><br />
Model #: LPG12-100<br><br />
Power Rating: 100 Ah/1200 Whr<br><br />
Voltage: 12 V<br><br />
Dimensions: 13” x 6.8” x 8.4”<br><br><br />
The battery is connected to the Arduino and the pump.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/54/CORNELL2014_Solarpanel2.JPG/800px-CORNELL2014_Solarpanel2.JPG" style="height: 250px;"><br />
<div class="caption"><br />
<b>Solar panel<br></b><br />
15 W mono-crystalline solar panel<br><br />
Model #: SPCC-15W <br><br><br />
The solar panel is used to recharge the battery.<br />
</div><br />
</a><br />
<a href="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" data-toggle="lightbox" class="thumbnail"> <br />
<img src="https://static.igem.org/mediawiki/2014/thumb/5/51/CORNELL2014_Foam.JPG/800px-CORNELL2014_Foam.JPG" style="height: 254px;"><br />
<div class="caption"><br />
<b>Foam and Acrylic for keeping components fixed within Pelican Case</b><br><br />
This padding material will hold items like the battery and filters in place and elevate them to an easily accessible area for maintenance. Grooves have been carved in the material to enable for easy monitoring of piping. <br />
</div><br />
</a><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/nickelTeam:Cornell/project/wetlab/nickel2014-10-18T02:21:38Z<p>E.Holmes: </p>
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<h1>Construct Design</h1><br />
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>.<br />
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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. <br />
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<img src="https://static.igem.org/mediawiki/2014/4/4f/Cornell_NixAc.png"><br />
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<h3>BBa_K1460006</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>nixA</i> should be transporting more nickel ions past the cell wall. This would lead to increased nickel sensitivity. To test for nickel sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460003 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with 1 mM Ni. <br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/9/94/Cornell_BL21_and_nixA_growth.png"><br />
<img src="https://static.igem.org/mediawiki/2014/9/94/Cornell_BL21_and_nixA_growth.png"><br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/1/15/Cornell_BL21_in_Ni.png"><br />
<img src="https://static.igem.org/mediawiki/2014/1/15/Cornell_BL21_in_Ni.png"><br />
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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 <i>nixA</i> 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.<br />
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Part BBa_K1460003 in pUC57 was co-transformed with part <a href="https://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. <br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/1/1f/Cornell_final_nickel_concentration.png"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/Cornell_final_nickel_concentration.png"><br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/c/ca/Cornell_nickel_per_OD.png"><br />
<img src="https://static.igem.org/mediawiki/2014/c/ca/Cornell_nickel_per_OD.png"><br />
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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. <br />
<br><br><br />
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="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.<br />
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<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Fulkerson, J., & Mobley, H. (2000). Membrane Topology of the <i>nixA</i> Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III. Journal of Bacteriology, 1722-1730.</li><br />
<li>Mobley, H., Garner, R., & Bauerfeind, P. (1995). Helicobacter pylori nickel-transport gene <i>nixA</i>: Synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Molecular Microbiology, 97-109.</li><br />
<li>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.</li><br />
</ol><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/mercuryTeam:Cornell/project/wetlab/mercury2014-10-18T02:20:54Z<p>E.Holmes: </p>
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<h1>Construct Design</h1><br />
To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells. <br />
<br><br><br />
Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-<i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. <br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Cornell_Mercury_crop.png"><br />
<div class="caption"><br />
<h3>BBa_K1460007</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). <br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/8/82/Cornell_merTandmerP_growth.png"><br />
<img src="https://static.igem.org/mediawiki/2014/8/82/Cornell_merTandmerP_growth.png"><br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/1/1c/Cornell_merandmet_growth.png"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1c/Cornell_merandmet_growth.png"> <br />
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What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. <br />
<br><br><br><br><br />
Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury 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.<br />
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There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and GST-<i>crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.</li><br />
<li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.</li><br />
</ol><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/leadTeam:Cornell/project/wetlab/lead2014-10-18T02:20:13Z<p>E.Holmes: </p>
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<h1>Construct Design</h1> <br />
In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene <i>cpb4</i> codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of <i>Bacillus spp.</i> found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant <i>Nicotiana tabacum</i>. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications <sup>[1]</sup>.<br />
<br><br><br />
Our first lead transporter construct <a href="http://parts.igem.org/Part:BBa_K1460005">(BBa_K1460005)</a> consists of the constitutive Anderson promoter and the <i>cbp4</i> gene for constitutive expression of the heavy metal membrane transporter and uptake of lead. The primary construct for lead sequestration <a href="http://parts.igem.org/Part:BBa_K1460008">(BBa_K1460008)</a> consists of this first lead transporter construct put upstream of our metallothionein construct with the <i>T7</i> promoter and <i>GST-crs5</i>. This construct allows for the constitutive expression of the lead transporter as well as the inducible expression of the metallothionein in <i>BL21</i> by arabinose activating the <i>araBAD</i> promoter and allowing expression of the highly active T7 polymerase. This allows for our bacterial strains to grow to stationary phase before being induced to produce metallothioneins and being used to sequester lead.<br />
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<h3>BBa_K1460008</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>cbp4</i> should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460005 in the amp<sup>r</sup> plasmid pUC57 were grown for a 24 hour period in LB with 1 mM Pb.<br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/f/f3/Cornell_leadgrowthgraph.png"><br />
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What we see after 24 hours of growth is no significant difference in growth between the two strains (figure 1). However, what we consistently observed is that there is no inhibition of growth of BL21 at high concentrations of lead (figure 2). Even if <i>cpb4</i> is expressed and is actively transporting lead ions into cells, it is possible that the concentration of lead is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test lead concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the lead nitrate that we were using for testing. <br />
<br><br> <br />
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Part BBa_K1460005 in pUC57 was co-transformed with part <a href="https://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 lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead 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. <br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/b/bf/Cornell_final_lead_concentration.png"><br />
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The chart on the left shows the average final concentration of lead in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 (figure 3). 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. This data suggests that cells engineered with <i>cbp4</i> and GST-<i>crs5</i> are in fact able to remove lead ions from water, and to the best of our knowledge this is the <b>first</b> successful bacterial lead sequestration system involving transport proteins and metallothioneins. <br />
<br><br><br />
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 as, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth. <br />
</div><br />
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<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
</ol><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlab/leadTeam:Cornell/project/wetlab/lead2014-10-18T02:18:42Z<p>E.Holmes: </p>
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<h1>Construct Design</h1> <br />
In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene <i>cpb4</i> codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of <i>Bacillus spp.</i> found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant <i>Nicotiana tabacum</i>. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications <sup>[1]</sup>.<br />
<br><br><br />
Our first lead transporter construct <a href="http://parts.igem.org/Part:BBa_K1460005">(BBa_K1460005)</a> consists of the constitutive Anderson promoter and the <i>cbp4</i> gene for constitutive expression of the heavy metal membrane transporter and uptake of lead. The primary construct for lead sequestration <a href="http://parts.igem.org/Part:BBa_K1460008">(BBa_K1460008)</a> consists of this first lead transporter construct put upstream of our metallothionein construct with the <i>T7</i> promoter and <i>GST-crs5</i>. This construct allows for the constitutive expression of the lead transporter as well as the inducible expression of the metallothionein in <i>BL21</i> by arabinose activating the <i>araBAD</i> promoter and allowing expression of the highly active T7 polymerase. This allows for our bacterial strains to grow to stationary phase before being induced to produce metallothioneins and being used to sequester lead.<br />
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<div class="col-md-12 col-xs-18"> <br />
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<img src="https://static.igem.org/mediawiki/2014/b/b0/Cornell_LeadPlasmid.png"><br />
<div class="caption"><br />
<h3>BBa_K1460008</h3><br />
</div><br />
</div><br />
</div><br />
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<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Results</h1><br />
Cells successfully expressing <i>cbp4</i> should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460005 in the amp<sup>r</sup> plasmid pUC57 were grown for a 24 hour period in LB with 1 mM Pb.<br />
</div><br />
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<div class="col-md-6 col-xs-9"> <br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/f/f3/Cornell_leadgrowthgraph.png"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f3/Cornell_leadgrowthgraph.png"><br />
</a><br />
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<div class="col-md-6 col-xs-9"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/6/6e/Cornell_BL21growthinlead.png"><br />
<img src="https://static.igem.org/mediawiki/2014/6/6e/Cornell_BL21growthinlead.png"><br />
</a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
What we see after 24 hours of growth is no significant difference in growth between the two strains (figure 1). However, what we consistently observed is that there is no inhibition of growth of BL21 at high concentrations of lead (figure 2). Even if <i>cpb4</i> is expressed and is actively transporting lead ions into cells, it is possible that the concentration of lead is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test lead concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the lead nitrate that we were using for testing. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
Part BBa_K1460005 in pUC57 was co-transformed with part <a href="https://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 lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead 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. <br />
</div><br />
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<div class="col-md-6 col-xs-9"><br />
<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/b/bf/Cornell_final_lead_concentration.png"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/Cornell_final_lead_concentration.png"><br />
</a><br />
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The chart on the left shows the average final concentration of lead in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 (figure 3). 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. This data suggests that cells engineered with <i>cbp4</i> and GST-<i>crs5</i> are in fact able to remove lead ions from water, and to the best of our knowledge this is the <b>first</b> successful bacterial lead sequestration system involving transport proteins and metallothioneins. <br />
<br><br><br />
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 as, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth.<br />
<br><br> <br />
</div><br />
</div><br />
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<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
</ol><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/hpracTeam:Cornell/project/hprac2014-10-18T02:16:19Z<p>E.Holmes: </p>
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<h1>Human Practices</h1> <br />
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Cornell iGEM Human Practices came into the year with much potential. Over the course of the past spring, summer, and fall we developed significant personal and academic investments in the subjects our team was tackling as a whole.<br />
<br><br><br />
We set out to create Human Practices components that contributed to and complemented with the work our team was doing, had a meaningful impact on our local and global communities, and were innovative, novel, and educational to future teams. To this end, we did the following: (1) engaged in extensive <a href="https://2014.igem.org/Team:Cornell/outreach">outreach</a>, (2) learned about the environmental, social, economic, and political issues that shaped the world of the biochemistry we were tackling, (3) launched a new social media platform called <a href="https://2014.igem.org/Team:Cornell/project/hprac/humans">Humans and SynBio</a> in collaboration with teams from across the world, (4) put together a <a href="https://2014.igem.org/Team:Cornell/project/hprac/survey">survey</a> to understand the constructs underlying opinions about synthetic biology, (5) built a <a href="https://2014.igem.org/Team:Cornell/project/hprac/ethics>Comprehensive Environmental Assessment</a>, following up on our efforts from previous years, (6) facilitated collaborations within our university to put together a <a href="https://2014.igem.org/Team:Cornell/project/futureapp">portfolio</a> of possible implementation of our genetically engineered technologies, (7) reached out to other iGEM teams to collect <a href="https://2014.igem.org/Team:Cornell/project/hprac/environ">water samples</a> for testing, and (8) considered the bioethical and safety implications of our work at large. <br />
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<h1>Humans and SynBio </h1><br />
This year we aimed to include an HPrac component that had a global impact, was adaptable, and served to educate both iGEM teams and the communities in which they operated, enhancing their relationships with each other. To this end, we took inspiration from the popular photoblog Humans of New York, which chronicles the personalities, visages, and life experiences of the people of New York City. HONY, as it’s called, has gained a worldwide following and has spawned numerous spin-off projects, including Humans of Ithaca and Humans of Cornell University. We sought to emulate HONY’s singular style, a mode of social media posting that is informative, striking, and familiar: every picture includes as its point of focus a person or group of people, and is accompanied by a quote from their conversation with the photographer, a piece of text that often highlights some unique quality of the interviewees.<br />
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For our project, we built a <a href="http://www.facebook.com/HumansandSynBio">Facebook page</a>. We produced a <a href="https://static.igem.org/mediawiki/2014/b/b6/Humans_and_Synbio_Invitation_-_Cornell_iGEM.pdf">document</a> that invited iGEM teams from across the world to contribute posts. This invitation outlines interview protocols, instructions for obtaining permission to post an interview transcript and photo online, and how the project relates to the broader goals shared by the iGEM competition and its constituent teams.<br />
<br><br><br />
After e-mailing this to all teams whose e-mails were readily available, as well as posting our invitation on the iGEM Facebook group several times this summer, results started to flow in. The submissions weren’t the only memorable element of this outreach - we learned a great deal about how individuals around the world think about and relate to synthetic biology.<br />
<br><br><br />
We continue to actively solicit and accept submissions for Humans & Synbio. Please contact us through Facebook if you are interested in participating!<br />
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<h1>SynBio Opinions </h1><br />
We surveyed a sample of our colleagues, peers, and community members (n=166), hoping to understand how individuals’ opinions about environmental issues and about the viability of synthetic biology affected their stated judgement of our synthetic biology application. We disseminated this survey using Facebook, e-mail, and other forms of social media. We also sent out invitations to all the iGEM teams who had their contact e-mails readily available on their websites. Results are summarized and pictured on the corresponding page, accompanied by a sample survey. Of note is the fact that out of the respondents who provided a complete set of responses (n=162), a distinct minority (n=3) indicated that they either disagreed or strongly disagreed (on a 5-point Likert scale) with the use of synthetic biology to implement the following description of our project:<br />
<br><br><br />
"This year Cornell iGEM will be focused on developing an alternative solution to heavy metal water pollution (i.e. lead, mercury, or nickel). Our hope is to create a water filtration device composed of <i>E. coli</i> that have been genetically engineered to produce metallothioneins - a protein that has a high affinity for binding with heavy metals. In other words, water containing heavy metals will be pumped through the <i>E. coli</i> cells and the heavy metals will be taken out of the water and into the <i>E. coli</i> cells. Our hope is to design our device for point-source filtration, so attaching it to the end of a factory pipe filtering out heavy metal content before it enters the ecosystem. However, there are many other applications for our project." <br />
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<h1>Environmental Water Samples </h1><br />
Instead of solely analyzing water samples from our area (Fall Creek in Ithaca, NY), we were curious to see how many other areas around the United States had traces of heavy metal contamination. Thus, we sent out a request for other iGEM teams to send us environmental water samples from their areas. We got responses from all across the nation, ranging from California, Utah, Michigan, Indiana, and Connecticut. In return, we analyzed their samples via ICP-AES (Inductively Coupled Plasma Atomic Emission Spectromy) and generated an individual water quality report for each team. Our goal was to develop a better understanding of heavy water contamination in drinking water in the United States, and the analyses returned a surprising variety of heavy metal concentrations in environmental water samples. <br />
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<h1>Risk Assessment </h1><br />
As engineers not only do we strive to design and create, we must ensure that whatever our product, it is safe for use, production, and marketing. In addition, we analyzed risk for community, the organism, the environment, and industries. In total, we conducted three different approaches to our risk assessment for Lead it Go. The first was developed by Cornell’s Environmental Health & Safety Department, pertaining specifically to work with recombinant organisms and the possible ramifications if they were to be released into the wild. The next, CEA (Comprehensive Environmental Assessment) was developed by the Environmental Protection Agency as a general environmental risk assessment and modified by both the Woodrow Wilson Center and our team for use on our synthetic biology project. Finally, we strived to embody the design principles set forth by the Presidential Commission for the Study of Bioethical Issues, to implement synthetic biology for the betterment of humanity. Each approach has its limitations, but all of them have helped to inform our project design, research practices, and considerations for further development of our project.<br />
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<h1>Human Practices</h1> <br />
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<div class="col-md-9 col-xs-12"><br />
Cornell iGEM Human Practices came into the year with much potential. Over the course of the past spring, summer, and fall we developed significant personal and academic investments in the subjects our team was tackling as a whole.<br />
<br><br><br />
We set out to create Human Practices components that contributed to and complemented with the work our team was doing, had a meaningful impact on our local and global communities, and were innovative, novel, and educational to future teams. To this end, we did the following: (1) engaged in extensive <a href="https://2014.igem.org/Team:Cornell/outreach">outreach</a>, (2) learned about the environmental, social, economic, and political issues that shaped the world of the biochemistry we were tackling, (3) launched a new social media platform called <a href="https://2014.igem.org/Team:Cornell/project/hprac/humans">Humans and SynBio</a> in collaboration with teams from across the world, (4) put together a <a href="https://2014.igem.org/Team:Cornell/project/hprac/survey">survey</a> to understand the constructs underlying opinions about synthetic biology, (5) built a <a href="https://2014.igem.org/Team:Cornell/project/hprac/ethics>Comprehensive Environmental Assessment</a>, following up on our efforts from previous years, (6) facilitated collaborations within our university to put together a <a href="https://2014.igem.org/Team:Cornell/project/futureapp">portfolio</a> of possible implementation of our genetically engineered technologies, (7) reached out to other iGEM teams to collect <a href="https://2014.igem.org/Team:Cornell/project/hprac/environ">water samples</a> for testing, and (8) considered the bioethical and safety implications of our work at large. <br />
</div><br />
<div class = "col-md-3 col-xs-6"> <br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Cornell_HASBprofile.png"><br />
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</div><br />
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<h1>Humans and SynBio </h1><br />
This year we aimed to include an HPrac component that had a global impact, was adaptable, and served to educate both iGEM teams and the communities in which they operated, enhancing their relationships with each other. To this end, we took inspiration from the popular photoblog Humans of New York, which chronicles the personalities, visages, and life experiences of the people of New York City. HONY, as it’s called, has gained a worldwide following and has spawned numerous spin-off projects, including Humans of Ithaca and Humans of Cornell University. We sought to emulate HONY’s singular style, a mode of social media posting that is informative, striking, and familiar: every picture includes as its point of focus a person or group of people, and is accompanied by a quote from their conversation with the photographer, a piece of text that often highlights some unique quality of the interviewees.<br />
</div><br />
<div class="row"><br />
<div class = "col-md-12 col-xs-18"> <br />
For our project, we built a <a href="http://www.facebook.com/HumansandSynBio">Facebook page</a>. We produced a <a href="https://static.igem.org/mediawiki/2014/b/b6/Humans_and_Synbio_Invitation_-_Cornell_iGEM.pdf">document</a> that invited iGEM teams from across the world to contribute posts. This invitation outlines interview protocols, instructions for obtaining permission to post an interview transcript and photo online, and how the project relates to the broader goals shared by the iGEM competition and its constituent teams.<br />
<br><br><br />
After e-mailing this to all teams whose e-mails were readily available, as well as posting our invitation on the iGEM Facebook group several times this summer, results started to flow in. The submissions weren’t the only memorable element of this outreach - we learned a great deal about how individuals around the world think about and relate to synthetic biology.<br />
<br><br><br />
We continue to actively solicit and accept submissions for Humans & Synbio. Please contact us through Facebook if you are interested in participating!<br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>SynBio Opinions </h1><br />
We surveyed a sample of our colleagues, peers, and community members (n=166), hoping to understand how individuals’ opinions about environmental issues and about the viability of synthetic biology affected their stated judgement of our synthetic biology application. We disseminated this survey using Facebook, e-mail, and other forms of social media. We also sent out invitations to all the iGEM teams who had their contact e-mails readily available on their websites. Results are summarized and pictured on the corresponding page, accompanied by a sample survey. Of note is the fact that out of the respondents who provided a complete set of responses (n=162), a distinct minority (n=3) indicated that they either disagreed or strongly disagreed (on a 5-point Likert scale) with the use of synthetic biology to implement the following description of our project:<br />
<br><br><br />
"This year Cornell iGEM will be focused on developing an alternative solution to heavy metal water pollution (i.e. lead, mercury, or nickel). Our hope is to create a water filtration device composed of <i>E. coli</i> that have been genetically engineered to produce metallothioneins - a protein that has a high affinity for binding with heavy metals. In other words, water containing heavy metals will be pumped through the <i>E. coli</i> cells and the heavy metals will be taken out of the water and into the <i>E. coli</i> cells. Our hope is to design our device for point-source filtration, so attaching it to the end of a factory pipe filtering out heavy metal content before it enters the ecosystem. However, there are many other applications for our project." <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Environmental Water Samples </h1><br />
Instead of solely analyzing water samples from our area (Fall Creek in Ithaca, NY), we were curious to see how many other areas around the United States had traces of heavy metal contamination. Thus, we sent out a request for other iGEM teams to send us environmental water samples from their areas. We got responses from all across the nation, ranging from California, Utah, Michigan, Indiana, and Connecticut. In return, we analyzed their samples via ICP-AES (Inductively Coupled Plasma Atomic Emission Spectromy) and generated an individual water quality report for each team. Our goal was to develop a better understanding of heavy water contamination in drinking water in the United States, and the analyses returned a surprising variety of heavy metal concentrations in environmental water samples. <br />
<br />
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<h1>Risk Assessment </h1><br />
</div> <br />
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</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/hpracTeam:Cornell/project/hprac2014-10-18T02:08:05Z<p>E.Holmes: </p>
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<h1>Human Practices</h1> <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-9 col-xs-12"><br />
Cornell iGEM Human Practices came into the year with much potential. Over the course of the past spring, summer, and fall we developed significant personal and academic investments in the subjects our team was tackling as a whole.<br />
<br><br><br />
We set out to create Human Practices components that contributed to and complemented with the work our team was doing, had a meaningful impact on our local and global communities, and were innovative, novel, and educational to future teams. To this end, we did the following: (1) engaged in extensive <a href="https://2014.igem.org/Team:Cornell/outreach">outreach</a>, (2) learned about the environmental, social, economic, and political issues that shaped the world of the biochemistry we were tackling, (3) launched a new social media platform called <a href="https://2014.igem.org/Team:Cornell/project/hprac/humans">Humans and SynBio</a> in collaboration with teams from across the world, (4) put together a <a href=https://2014.igem.org/Team:Cornell/project/hprac/survey">survey</a> to understand the constructs underlying opinions about synthetic biology, (5) built a <a href="https://2014.igem.org/Team:Cornell/project/hprac/ethics>Comprehensive Environmental Assessment</a>, following up on our efforts from previous years, (6) facilitated collaborations within our university to put together a <a href="https://2014.igem.org/Team:Cornell/project/futureapp">portfolio</a> of possible implementation of our genetically engineered technologies, (7) reached out to other iGEM teams to collect <a href="https://2014.igem.org/Team:Cornell/project/hprac/environ">water samples</a> for testing, and (8) considered the bioethical and safety implications of our work at large. <br />
</div><br />
<div class = "col-md-3 col-xs-6"> <br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Cornell_HASBprofile.png"><br />
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<img src="https://static.igem.org/mediawiki/2014/a/a5/Cornell_humans18.jpg"><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Humans and SynBio </h1><br />
This year we aimed to include an HPrac component that had a global impact, was adaptable, and served to educate both iGEM teams and the communities in which they operated, enhancing their relationships with each other. To this end, we took inspiration from the popular photoblog Humans of New York, which chronicles the personalities, visages, and life experiences of the people of New York City. HONY, as it’s called, has gained a worldwide following and has spawned numerous spin-off projects, including Humans of Ithaca and Humans of Cornell University. We sought to emulate HONY’s singular style, a mode of social media posting that is informative, striking, and familiar: every picture includes as its point of focus a person or group of people, and is accompanied by a quote from their conversation with the photographer, a piece of text that often highlights some unique quality of the interviewees.<br />
</div><br />
<div class="row"><br />
<div class = "col-md-12 col-xs-18"> <br />
For our project, we built a <a href="http://www.facebook.com/HumansandSynBio">Facebook page</a>. We produced a <a href="https://static.igem.org/mediawiki/2014/b/b6/Humans_and_Synbio_Invitation_-_Cornell_iGEM.pdf">document</a> that invited iGEM teams from across the world to contribute posts. This invitation outlines interview protocols, instructions for obtaining permission to post an interview transcript and photo online, and how the project relates to the broader goals shared by the iGEM competition and its constituent teams.<br />
<br><br><br />
After e-mailing this to all teams whose e-mails were readily available, as well as posting our invitation on the iGEM Facebook group several times this summer, results started to flow in. The submissions weren’t the only memorable element of this outreach - we learned a great deal about how individuals around the world think about and relate to synthetic biology.<br />
<br><br><br />
We continue to actively solicit and accept submissions for Humans & Synbio. Please contact us through Facebook if you are interested in participating!<br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>SynBio Opinions </h1><br />
We surveyed a sample of our colleagues, peers, and community members (n=166), hoping to understand how individuals’ opinions about environmental issues and about the viability of synthetic biology affected their stated judgement of our synthetic biology application. We disseminated this survey using Facebook, e-mail, and other forms of social media. We also sent out invitations to all the iGEM teams who had their contact e-mails readily available on their websites. Results are summarized and pictured on the corresponding page, accompanied by a sample survey. Of note is the fact that out of the respondents who provided a complete set of responses (n=162), a distinct minority (n=3) indicated that they either disagreed or strongly disagreed (on a 5-point Likert scale) with the use of synthetic biology to implement the following description of our project:<br />
<br><br><br />
"This year Cornell iGEM will be focused on developing an alternative solution to heavy metal water pollution (i.e. lead, mercury, or nickel). Our hope is to create a water filtration device composed of <i>E. coli</i> that have been genetically engineered to produce metallothioneins - a protein that has a high affinity for binding with heavy metals. In other words, water containing heavy metals will be pumped through the <i>E. coli</i> cells and the heavy metals will be taken out of the water and into the <i>E. coli</i> cells. Our hope is to design our device for point-source filtration, so attaching it to the end of a factory pipe filtering out heavy metal content before it enters the ecosystem. However, there are many other applications for our project." <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Environmental Water Samples </h1><br />
Instead of solely analyzing water samples from our area (Fall Creek in Ithaca, NY), we were curious to see how many other areas around the United States had traces of heavy metal contamination. Thus, we sent out a request for other iGEM teams to send us environmental water samples from their areas. We got responses from all across the nation, ranging from California, Utah, Michigan, Indiana, and Connecticut. In return, we analyzed their samples via ICP-AES (Inductively Coupled Plasma Atomic Emission Spectromy) and generated an individual water quality report for each team. Our goal was to develop a better understanding of heavy water contamination in drinking water in the United States, and the analyses returned a surprising variety of heavy metal concentrations in environmental water samples. <br />
<br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Risk Assessment </h1><br />
</div> <br />
<br />
</div><br />
<br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/hpracTeam:Cornell/project/hprac2014-10-18T02:04:14Z<p>E.Holmes: </p>
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<h1>Human Practices</h1> <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-9 col-xs-12"><br />
Cornell iGEM Human Practices came into the year with much potential. Over the course of the past spring, summer, and fall we developed significant personal and academic investments in the subjects our team was tackling as a whole.<br />
<br><br><br />
We set out to create Human Practices components that contributed to and complemented with the work our team was doing, had a meaningful impact on our local and global communities, and were innovative, novel, and educational to future teams. To this end, we did the following: (1) engaged in extensive outreach, (2) learned about the environmental, social, economic, and political issues that shaped the world of the biochemistry we were tackling, (3) launched a new social media platform called Humans and SynBio in collaboration with teams from across the world, (4) put together a survey to understand the constructs underlying opinions about synthetic biology, (5) built a Comprehensive Environmental Assessment, following up on our efforts from previous years, (6) facilitated collaborations within our university to put together a <a href="https://2014.igem.org/Team:Cornell/project/futureapp">portfolio</a> of possible implementation of our genetically engineered technologies, (7) reached out to other iGEM teams to collect water samples for testing, and (8) considered the bioethical and safety implications of our work at large. <br />
</div><br />
<div class = "col-md-3 col-xs-6"> <br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f6/Cornell_HASBprofile.png"><br />
</div><br />
</div><br />
</div> <br />
<div class="row"><br />
<div class = "col-md-4 col-xs-6"> <br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/a/a5/Cornell_humans18.jpg"><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-12"><br />
<h1>Humans and SynBio </h1><br />
This year we aimed to include an HPrac component that had a global impact, was adaptable, and served to educate both iGEM teams and the communities in which they operated, enhancing their relationships with each other. To this end, we took inspiration from the popular photoblog Humans of New York, which chronicles the personalities, visages, and life experiences of the people of New York City. HONY, as it’s called, has gained a worldwide following and has spawned numerous spin-off projects, including Humans of Ithaca and Humans of Cornell University. We sought to emulate HONY’s singular style, a mode of social media posting that is informative, striking, and familiar: every picture includes as its point of focus a person or group of people, and is accompanied by a quote from their conversation with the photographer, a piece of text that often highlights some unique quality of the interviewees.<br />
</div><br />
<div class="row"><br />
<div class = "col-md-12 col-xs-18"> <br />
For our project, we built a <a href="http://www.facebook.com/HumansandSynBio">Facebook page</a>. We produced a <a href="https://static.igem.org/mediawiki/2014/b/b6/Humans_and_Synbio_Invitation_-_Cornell_iGEM.pdf">document</a> that invited iGEM teams from across the world to contribute posts. This invitation outlines interview protocols, instructions for obtaining permission to post an interview transcript and photo online, and how the project relates to the broader goals shared by the iGEM competition and its constituent teams.<br />
<br><br><br />
After e-mailing this to all teams whose e-mails were readily available, as well as posting our invitation on the iGEM Facebook group several times this summer, results started to flow in. The submissions weren’t the only memorable element of this outreach - we learned a great deal about how individuals around the world think about and relate to synthetic biology.<br />
<br><br><br />
We continue to actively solicit and accept submissions for Humans & Synbio. Please contact us through Facebook if you are interested in participating!<br />
</div><br />
</div> <br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>SynBio Opinions </h1><br />
We surveyed a sample of our colleagues, peers, and community members (n=166), hoping to understand how individuals’ opinions about environmental issues and about the viability of synthetic biology affected their stated judgement of our synthetic biology application. We disseminated this survey using Facebook, e-mail, and other forms of social media. We also sent out invitations to all the iGEM teams who had their contact e-mails readily available on their websites. Results are summarized and pictured on the corresponding page, accompanied by a sample survey. Of note is the fact that out of the respondents who provided a complete set of responses (n=162), a distinct minority (n=3) indicated that they either disagreed or strongly disagreed (on a 5-point Likert scale) with the use of synthetic biology to implement the following description of our project:<br />
<br><br><br />
"This year Cornell iGEM will be focused on developing an alternative solution to heavy metal water pollution (i.e. lead, mercury, or nickel). Our hope is to create a water filtration device composed of <i>E. coli</i> that have been genetically engineered to produce metallothioneins - a protein that has a high affinity for binding with heavy metals. In other words, water containing heavy metals will be pumped through the <i>E. coli</i> cells and the heavy metals will be taken out of the water and into the <i>E. coli</i> cells. Our hope is to design our device for point-source filtration, so attaching it to the end of a factory pipe filtering out heavy metal content before it enters the ecosystem. However, there are many other applications for our project." <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Environmental Water Samples </h1><br />
Instead of solely analyzing water samples from our area (Fall Creek in Ithaca, NY), we were curious to see how many other areas around the United States had traces of heavy metal contamination. Thus, we sent out a request for other iGEM teams to send us environmental water samples from their areas. We got responses from all across the nation, ranging from California, Utah, Michigan, Indiana, and Connecticut. In return, we analyzed their samples via ICP-AES (Inductively Coupled Plasma Atomic Emission Spectromy) and generated an individual water quality report for each team. Our goal was to develop a better understanding of heavy water contamination in drinking water in the United States, and the analyses returned a surprising variety of heavy metal concentrations in environmental water samples. <br />
<br />
</div> <br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Risk Assessment </h1><br />
</div> <br />
<br />
</div><br />
<br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T02:01:25Z<p>E.Holmes: </p>
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
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<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
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<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h4> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Water Filters:</b><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br><br />
<b>Redox media, non-chemical water treatment:</b><br />
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>[15]</sup>. <br />
<br><br />
<b>Guar Gum:</b> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
<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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T02:00:51Z<p>E.Holmes: </p>
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h4> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Water Filters:</b><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br><br />
<b>Redox media, non-chemical water treatment:</b><br />
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>[15]</sup>. <br />
<br><br />
<b>Guar Gum:</b> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
<li>[12] 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><br />
<li>[13] 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><br />
<li>[14] 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><br />
<li>[15] 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><br />
<li>[16] 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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:56:59Z<p>E.Holmes: </p>
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h4> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Water Filters:</b><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br><br />
<b>Redox media, non-chemical water treatment:</b><br />
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>[15]</sup>. <br />
<br><br />
<b>Guar Gum:</b> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:55:20Z<p>E.Holmes: </p>
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{{:Team:Cornell/project/background/header}}<br />
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$(window).load(function() {<br />
$('li.p_back_lead').addClass('active');<br />
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h4> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Water Filters:</b><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br />
<b>Redox media, non-chemical water treatment:</b><br />
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>[15]</sup>. <br />
<br />
<b>Guar Gum:</b> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:54:53Z<p>E.Holmes: </p>
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{{:Team:Cornell/project/background/header}}<br />
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$(window).load(function() {<br />
$('li.p_back_lead').addClass('active');<br />
});<br />
</script><br />
<body><br />
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<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h4> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<b>Water Filters:</b><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br />
<b>Redox media, non-chemical water treatment:<b><br />
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>[15]</sup>. <br />
<br />
<b>Guar Gum:<b> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:53:55Z<p>E.Holmes: </p>
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h3> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<h4>Water Filters:</h4><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br />
<h4>Redox media, non-chemical water treatment:</h4><br />
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>[15]</sup>. <br />
<br />
<h4>Guar Gum:</h4> Adsorption by this unique compound<sup>[16]</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:53:23Z<p>E.Holmes: </p>
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$('li.p_back_lead').addClass('active');<br />
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<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h3> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
<h4>Water Filters:</h4><br />
<br><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
<br />
<h4>Redox media, non-chemical water treatment:</h4><br />
<br><br />
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>[15]</sup>. <br />
<br />
<h4>Guar Gum:</h4> Adsorption by this unique compound<sup>[16}</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.<br />
<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:52:52Z<p>E.Holmes: </p>
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<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
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<b>Extreme cases of high lead poisoning</b><br />
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<li>Neurological damage</li><br />
<li>Death</li><br />
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<h1>Case Studies</h1><br />
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><br />
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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>. <br />
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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><br />
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<h4>Ithaca Gun Factory:</h3> <br />
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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 [11]. 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. <br />
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The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
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<h1>Current Remediation Techniques</h1><br />
<h3>Water Filters:</h3><br />
<br><br />
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>[12]</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>[13]</sup>. Increased water corrosivity, influenced by pH<sup>[14]</sup>, generally results in a higher lead content. <br />
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<h3>Redox media, non-chemical water treatment:</h3><br />
<br><br />
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>[15]</sup>. <br />
<br />
<h3>Guar Gum:</h3> Adsorption by this unique compound<sup>[16}</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.<br />
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<h1><i>cbp4</i></h1><br />
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.<br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
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<h1>Human Practices</h1> <br />
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Cornell iGEM Human Practices came into the year with much potential. Over the course of the past spring, summer, and fall we developed significant personal and academic investments in the subjects our team was tackling as a whole.<br />
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We set out to create Human Practices components that contributed to and complemented with the work our team was doing, had a meaningful impact on our local and global communities, and were innovative, novel, and educational to future teams. To this end, we did the following: (1) engaged in extensive outreach, (2) learned about the environmental, social, economic, and political issues that shaped the world of the biochemistry we were tackling, (3) launched a new social media platform called Humans and SynBio in collaboration with teams from across the world, (4) put together a survey to understand the constructs underlying opinions about synthetic biology, (5) built a Comprehensive Environmental Assessment, following up on our efforts from previous years, (6) facilitated collaborations within our university to put together a portfolio of possible implementation of our genetically engineered technologies, (7) reached out to other iGEM teams to collect water samples for testing, and (8) considered the bioethical and safety implications of our work at large. <br />
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<h1>Humans and SynBio </h1><br />
This year we aimed to include an HPrac component that had a global impact, was adaptable, and served to educate both iGEM teams and the communities in which they operated, enhancing their relationships with each other. To this end, we took inspiration from the popular photoblog Humans of New York, which chronicles the personalities, visages, and life experiences of the people of New York City. HONY, as it’s called, has gained a worldwide following and has spawned numerous spin-off projects, including Humans of Ithaca and Humans of Cornell University. We sought to emulate HONY’s singular style, a mode of social media posting that is informative, striking, and familiar: every picture includes as its point of focus a person or group of people, and is accompanied by a quote from their conversation with the photographer, a piece of text that often highlights some unique quality of the interviewees.<br />
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For our project, we built a <a href="http://www.facebook.com/HumansandSynBio">Facebook page</a>. We produced a <a href="https://static.igem.org/mediawiki/2014/b/b6/Humans_and_Synbio_Invitation_-_Cornell_iGEM.pdf">document</a> that invited iGEM teams from across the world to contribute posts. This invitation outlines interview protocols, instructions for obtaining permission to post an interview transcript and photo online, and how the project relates to the broader goals shared by the iGEM competition and its constituent teams.<br />
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After e-mailing this to all teams whose e-mails were readily available, as well as posting our invitation on the iGEM Facebook group several times this summer, results started to flow in. The submissions weren’t the only memorable element of this outreach - we learned a great deal about how individuals around the world think about and relate to synthetic biology.<br />
<br><br><br />
We continue to actively solicit and accept submissions for Humans & Synbio. Please contact us through Facebook if you are interested in participating!<br />
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<h1>SynBio Opinions </h1><br />
We surveyed a sample of our colleagues, peers, and community members (n=166), hoping to understand how individuals’ opinions about environmental issues and about the viability of synthetic biology affected their stated judgement of our synthetic biology application. We disseminated this survey using Facebook, e-mail, and other forms of social media. We also sent out invitations to all the iGEM teams who had their contact e-mails readily available on their websites. Results are summarized and pictured on the corresponding page, accompanied by a sample survey. Of note is the fact that out of the respondents who provided a complete set of responses (n=162), a distinct minority (n=3) indicated that they either disagreed or strongly disagreed (on a 5-point Likert scale) with the use of synthetic biology to implement the following description of our project:<br />
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"This year Cornell iGEM will be focused on developing an alternative solution to heavy metal water pollution (i.e. lead, mercury, or nickel). Our hope is to create a water filtration device composed of <i>E. coli</i> that have been genetically engineered to produce metallothioneins - a protein that has a high affinity for binding with heavy metals. In other words, water containing heavy metals will be pumped through the <i>E. coli</i> cells and the heavy metals will be taken out of the water and into the <i>E. coli</i> cells. Our hope is to design our device for point-source filtration, so attaching it to the end of a factory pipe filtering out heavy metal content before it enters the ecosystem. However, there are many other applications for our project." <br />
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<h1>Environmental Water Samples </h1><br />
Instead of solely analyzing water samples from our area (Fall Creek in Ithaca, NY), we were curious to see how many other areas around the United States had traces of heavy metal contamination. Thus, we sent out a request for other iGEM teams to send us environmental water samples from their areas. We got responses from all across the nation, ranging from California, Utah, Michigan, Indiana, and Connecticut. In return, we analyzed their samples via ICP-AES (Inductively Coupled Plasma Atomic Emission Spectromy) and generated an individual water quality report for each team. Our goal was to develop a better understanding of heavy water contamination in drinking water in the United States, and the analyses returned a surprising variety of heavy metal concentrations in environmental water samples. <br />
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<h1>Risk Assessment </h1><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/wetlabTeam:Cornell/project/wetlab2014-10-18T01:40:12Z<p>E.Holmes: </p>
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<h1>Idea</h1><br />
We sought to create genetically engineered strains of <i>E.coli</i> that could sequester the heavy metals nickel, mercury, and lead from water sources. Each strain included a metal transport protein specific to the respective metal as well as a metallothionein to bind metal ions intracellularly. We first generated basic, functional BioBricks containing: a glutathione-s-transferase gene and yeast metallothionein (<i>crs5</i>) gene fusion for metal binding <a href="http://parts.igem.org/Part:BBa_K1460001">(BBa_K1460001)</a><sup>1</sup>; the nickel transport gene <i>nixA</i> for selective nickel uptake <a href="http://parts.igem.org/Part:BBa_K1460003">(BBa_K1460003)</a><sup>2</sup>; the mercury transporter genes <i>merT</i> and <i>merP</i> for selective mercury uptake <a href="http://parts.igem.org/Part:BBa_K1460004">(BBa_K1460004)</a><sup>3</sup>; and a putative lead transport gene <i>cbp4</i> for selective lead uptake <a href="http://parts.igem.org/Part:BBa_K1460005">(BBa_K1460005)</a><sup>4</sup>. <br />
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<br><br />
To test for sequestration ability of combined systems, each transport protein in the amp<sup>r</sup> vector pUC57 was co-transformed with the metallothionein fusion in the cm<sup>r</sup> plasmid pSB1C3 and selected for using both markers. These strains are the functional equivalents of the BioBricks <a href="http://parts.igem.org/Part:BBa_K1460006">BBa_K1460006</a>, <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a>, and <a href="http://parts.igem.org/Part:BBa_K1460008">BBa_K1460008</a>, and these strains were tested for sequestration ability of nickel, mercury, and lead respectively. <br />
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In addition, the sequestering strains can placed into fiber reactors to develop functional sequestering filters (see <a href="https://2014.igem.org/Team:Cornell/project/drylab">dry lab)</a>.The idea of utilizing metallothioneins in parallel with metal transporters for sequestration has been studied for mercury and nickel but has never been explored for lead.<br />
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<h1>Components</h1><br />
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<h1>Experiments</h1><br />
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><br />
<ol><br />
<li> Spectrophotometer was used to analyze and compare the kinetic growth rates of <i>E. coli</i> cultures expressing only the metallothionein protein, only the transporter proteins, both metallothionein and transporter proteins, and just the vector backbone as a control. </li><br />
<ul><br />
<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><br />
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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><br />
<ul><br />
<li> Nutrient Analysis Lab at Cornell University using ICP-AES</li><br />
<li> Using green-fluorescent heavy metal indicator Phen Green</li><br />
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<h1>Results</h1><br />
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<li>BL21 strains with the <i>merT/merP</i> 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> <br />
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<li>When considering 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><br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<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><br />
<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><br />
<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><br />
<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. The Plant Journal, 171-182</li><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2014-10-18T01:36:30Z<p>E.Holmes: </p>
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<h1 style="padding: 0px; margin-bottom: 0px;">Safety</h1><br />
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While our project aims to help alleviate environmental pollution, we must take precautions to ensure that no personal or environmental harm comes in the process. Biological lab work comes with an inherent risk, and outlined below are specific risks associated with our project as well as precautions we take to safely complete our project. Our completed safety form can be found <a href="https://igem.org/Safety/Safety_Form?team_id=1460" target="_blank">here</a>.<br />
<h1 style="margin-top: 0px;">Specific Safety Concerns</h1><br />
<b>Laboratory Safety:</b><br />
To visualize DNA gels, we regularly use ethidium bromide, a DNA intercalating agent known to be carcinogenic. To visualize gels, we also use a powerful UV light. We regularly use the antibiotics chloramphenicol and ampicillin, which can be harmful to humans in large doses. For our project this year we are using salts of the heavy metals nickel, lead, and mercury. These metals can be acutely toxic at high enough concentrations and carcinogenic over the long term. We also use open flame alcohol burners to maintain a sterile environment. <br />
<br><br><br />
<b>Environmental Safety:</b><br />
If any biological materials escape from the lab there is a risk of transfer of antibiotic resistance from our engineered strains into other organisms. None of the coding regions we're using in our project should provide competitive advantage in a natural environment (unless in an environment with high metal concentrations), so the biggest risk is from transfer of antibiotic resistance to outside organisms. Additionally, release of the heavy metals we are working with this year (Ni, Hg, Pb) could also pose environmental and personal harm if released into the water supply. <br />
<br><br><br />
<b>Chassis Organisms:</b><br />
The table below describes all strains worked with for our project this year. The chassis organisms <i>E. coli</i> DH5a and BL21-AI are biosafety level 1 organisms and pose no disease risk to people. None of the coding regions we're using in our project should provide competitive advantage in a natural environment (unless in an environment with high metal concentrations), so the biggest risk is from transfer of antibiotic resistance to outside organisms.<br />
<br><br><br />
<table style="width:100%"> <br />
<tr><br />
<td>Species name</td><br />
<td>Risk Group</td> <br />
<td>Risk Group Source</td><br />
<td>Disease risk to humans?</td><br />
<td>Part number/name</td><br />
<td>Natural function of part</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli DH5α</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>N/A</td><br />
<td>N/A</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli</i> BL21-AI</td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>T7 polymerase</td><br />
<td>RNA polymerase native to T7 bacteriophage</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli BL21-AI</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460001</td><br />
<td>GST enzyme transfers reduced form of glutathione to xenobiotic substrates. YMT binds to metals to confer metal tolerance.</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli BL21-AI</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460002</td><br />
<td>GST enzyme transfers reduced form of glutathione to xenobiotic substrates. YMT binds to metals to confer metal tolerance.</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli DH5α</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460003</td><br />
<td>NixA transport nickel ions in <i>H. pylori</i></td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli DH5α</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460004</td><br />
<td>merT/merP transport mercury ions in <i>P. aeruginosa</i></td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli DH5α</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460005</td><br />
<td>CBP4 is used in <i>N. tabacum</i> to confer nickel tolerance and transport lead</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli BL21-AI</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460006</td><br />
<td>Composite Part</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli BL21-AI</i>/td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460007</td><br />
<td>Composite Part</td><br />
</tr><br />
<tr><br />
<td><i>Escherichia coli BL21-AI</i></td><br />
<td>1</td> <br />
<td>NIH</td><br />
<td>No</td><br />
<td>BBa_K1460008</td><br />
<td>Composite Part</td><br />
</tr> <br />
</table> <br />
<br />
<h1>Safety Protocol</h1><br />
<b>Wet lab:</b><br />
All lab members wear nitrile gloves, closed-toe shoes, and use eye protection when working with volatile chemicals or UV light. Gloves are replaced and hands are washed immediately after using ethidium bromide or any of the metal solutions. Members work in small groups to ensure if any harm comes to one, others are there to assist. When working with a new reagent or piece of equipment, a faculty lab manager or experienced member is always present to assist.<br />
<br><br><br />
There are taped off, designated areas for working with both ethidium bromide and the heavy metal solutions. These areas are cleaned before and after work and are the only areas these solutions may touch. All toxic waste is placed in a specialized receptacle and is picked up and disposed of by Cornell Environmental Health and Safety.<br />
<br><br><br />
All disposables that come in contact with biologics are disposed of in biohazard waste. The lab space also contains sharps containers for disposal of all sharps that contact biological material. All biohazard waste is autoclaved and transported to the building's centralized waste facility where it is disposed of as regulated biological waste.<br />
<br><br><br />
We maintain 2 copies of MSDS's for every chemical we use in the lab: one for our own records and one for the lab manager and users of the lab space who are not part of our team (list of all chemicals is attached as an excel document). The lab is equipped with flame-retardant benches, spill kits, safety showers, eye-washes, and fire extinguishers.<br />
<br><br><br />
<b>Dry lab:</b><br />
We use the Emerson Machine Shop for fabrication; each of the dry lab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the dry lab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed-toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects.<br />
<br />
<h1>Training and Enforcement:</h1><br />
<br />
<b>Training:</b><br />
All team members who work in the wet lab must complete Cornell EH&S general lab safety and chemical waste disposal courses prior to the onset of work. These courses set specific guidelines and are the standard requirement for work in a biosafety-level 1 lab at Cornell. Additionally, all team members must complete a lab orientation session with the manager of the BME instructional lab, Dr. Shivaun Archer. During these sessions, Dr. Archer familiarizes new members with the safety equipment and procedures specific to the labspace in which we work. <br />
<br><br><br />
Prior to the onset of work for the year, all new members are required to go through a 1 week training program. During this program, safety officers reinforce safety procedures learned during the EH&S courses, discuss safety protocol pertaining to specific chemicals with which we work, and ensure all lab members fully understand all safety procedures. <br />
<br><br><br />
<b>Safety Officers:</b><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu" target="_blank" >Institutional Biosafety Committee </a> to ensure proper equipment usage. <br />
<br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx" target="_blank">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly. <br />
<br><br><br />
<b>Enforcement:</b> <br />
Team members who violate safety rules are required to work under the supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of performing the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
<br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/background/leadTeam:Cornell/project/background/lead2014-10-18T01:32:23Z<p>E.Holmes: </p>
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h3> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
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<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
</div><br />
</div><br />
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<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
</div><br />
</div><br />
</div><br />
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<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
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<h1 style="margin-top: 0px;">Health Risks</h1><br />
Lead has no known biological 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.<br />
<br><br><br />
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.<br />
<br><br><br />
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>[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<sup>[8]</sup>. Lead has also been shown to inhibit hippocampal long-term potentiation, a neural mechanism required for learning<sup>[8]</sup>.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-6 col-xs-9"><br />
<b><font size=3>Side Effects of Lead Poisoning:</font></b><br />
<br><br><br />
<b>For infants and children:</b><br />
<ul><br />
<li>Impaired neurological development</li><br />
<li>Gastrointestinal distress</li><br />
<li>Anemia</li><br />
<li>Kidney failure</li><br />
<li>Irritability</li><br />
<li>Lethargy</li><br />
<li>Learning disabilities</li><br />
<li>Erratic behavior.</li><br />
</ul><br />
<b>For adults:</b><br />
<ul><br />
<li>Gastrointestinal distress</li><br />
<li>Weakness</li><br />
<li>Pins and needles</li><br />
<li>Kidney failure</li><br />
</ul><br />
<b>Extreme cases of high lead poisoning</b><br />
<ul><br />
<li>Neurological damage</li><br />
<li>Death</li><br />
</ul><br />
</div><br />
<div class="col-md-6 col-xs-9"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bd/Screen_Shot_2014-10-15_at_11.42.29_PM.png"><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Case Studies</h1><br />
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><br />
<br><br><br />
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>. <br />
<br><br><br />
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><br />
<br><br><br />
<h4>Ithaca Gun Factory:</h3> <br />
<br><br />
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 [11]. 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. <br />
<br />
The lead pollution seeped into the Cayuga Watershed and has been a common issue ever since [4]. 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. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1>Current Remediation Techniques</h1><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<div id="CBP4"><br />
<h1><i>cbp4</i></h1><br />
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.<br />
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<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<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><br />
<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><br />
<li>Eapen, S., & Dsouza, S. (2004). Prospects Of Genetic Engineering Of Plants For Phytoremediation Of Toxic Metals. Biotechnology Advances, 97-114.</li><br />
<li>"Public Health - Seattle & King County." Lead and Its Human Effects. King County Government, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Pathophysiology and Etiology of Lead Toxicity ." Pathophysiology and Etiology of Lead Toxicity. Medscape, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Consumer Factsheet on Lead in Drinking Water." Home. Environmental Protection Agency, n.d. Web. 15 Oct. 2014.</li><br />
<li>"Why Lead Used to Be Added To Gasoline." Today I Found Out RSS. N.p., n.d. Web. 15 Oct. 2014.</li><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/modelingTeam:Cornell/project/modeling2014-10-18T01:25:20Z<p>E.Holmes: </p>
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<h1>Effectiveness and economic feasibility of our hollow fiber bioreactor system</h1><br />
To determine the effectiveness and economic feasibility of our hollow fiber bioreactor with <br />
<i> E. coli</i> which has been engineered to express a mercury transport system and metallothionein, we modeled its impact when applied to a current real situation: mercury pollution in Onondaga Lake, Syracuse, NY.<br />
<br> <br><br />
It has been shown that similar hollow fiber bioreactors are able to reduce the concentration of mercury from 2mg/L to about 5 µg/L.<sup>[1]</sup> This corresponds to a promising 99.8% reduction in mercury levels. Furthermore as discussed in our case study, Onondaga Lake has a capacity of 35 billion gallons and about 165,000 lbs of mercury has been dumped into the lake over the years.[2] This corresponds to an approximate mercury concentration of 0.56 mg/L. Thus, the mercury concentration is Onondaga Lake is within the limits that the engineered <i> E. coli</i> is able to sequester. <br />
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<img src="https://static.igem.org/mediawiki/2014/e/e0/Cornell_Onondaga_Lake_Park.jpg" alt="Onondaga Lake Park: Syracuse, NY"><br />
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Onondaga Lake Park: Syracuse, NY<br />
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<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" alt="Hollow fiber bioreactor system"> <br />
<div class="caption center"><br />
Hollow fiber bioreactor system<br />
</div><br />
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In Nov 2004, the estimated cost of dredging to remove the mercury contaminated mud in the lake was determined to be $451 million.<sup>[3]</sup> Currently the cost of our hollow fiber bioreactor system is about $560 with the cost being largely due to the reactor itself ($490) and the remainder of the cost was for the pump and filters. <br />
<br> <br><br />
However, it should be noted that the scale of the hollow fiber bioreactor system is much smaller as its volume is about 1L. Hence, the hollow fiber bioreactor would have to be scaled up significantly (by about 10<sup>11</sup> times!) in order to have any impact. To give a better idea of the scale, if the lake were the size of an Olympic swimming pool, the volume of the hollow fiber bioreactor would be equivalent to a drop of water. While we would need to scale up the volume of our hollow fiber bioreactor, it should also be noted that by placing the bioreactors in series, better mercury sequestration is achieved.<sup>[1]</sup> <br />
Therefore, even though it has been shown that the hollow fiber bioreactor is successful on a pilot scale, more tests would be required to determine if it is as effective on a larger scale. As there are several variables that might change e.g. flow rates and membrane area, the performance of the engineered <i>E. coli</i> might not simply scale up as expected. Nevertheless, given the environmental costs associated with existing remediation methods such as dredging, it is important to look into how biological systems are able to complement these solutions and solve the problem of mercury contamination in an effective, safe and cost efficient manner. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Chen, S., Kim, E., Shuler, M., & Wilson, D. (1998). Hg2+ Removal by Genetically Engineered Escherichia coli in a Hollow Fiber Bioreactor. Biotechnology Progress, 667-671</li><br />
<li>Moriarty, Rick. "Discovering What Lies at the Bottom of Onondaga Lake." Syracuse.com. Syracuse.com, n.d. Web. 24 Sept. 2014.</li><br />
<li>Collin, Glen. "Onondaga Lake Dredging Begins for Season; Could End a Year Early (video)." Syracuse.com. N.p., 7 Apr. 2014. Web. 11 Aug. 2014.</li><br />
</ol><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/modelingTeam:Cornell/project/modeling2014-10-18T01:24:52Z<p>E.Holmes: </p>
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<div class="col-md-8 col-xs-15"><br />
<h1>Effectiveness and economic feasibility of our hollow fiber bioreactor system</h1><br />
To determine the effectiveness and economic feasibility of our hollow fiber bioreactor with <br />
<i> E. coli</i> which has been engineered to express a mercury transport system and metallothionein, we modeled its impact when applied to a current real situation, mercury pollution in Onondaga Lake, Syracuse, NY.<br />
<br> <br><br />
It has been shown that similar hollow fiber bioreactors are able to reduce the concentration of mercury from 2mg/L to about 5 µg/L.<sup>[1]</sup> This corresponds to a promising 99.8% reduction in mercury levels. Furthermore as discussed in our case study, Onondaga Lake has a capacity of 35 billion gallons and about 165,000 lbs of mercury has been dumped into the lake over the years.[2] This corresponds to an approximate mercury concentration of 0.56 mg/L. Thus, the mercury concentration is Onondaga Lake is within the limits that the engineered <i> E. coli</i> is able to sequester. <br />
</div><br />
<div class="col-md-4 col-xs-5"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e0/Cornell_Onondaga_Lake_Park.jpg" alt="Onondaga Lake Park: Syracuse, NY"><br />
<div class="caption center"><br />
Onondaga Lake Park: Syracuse, NY<br />
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</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-5"><br />
<div class="thumbnail" style="margin-top:0;"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" alt="Hollow fiber bioreactor system"> <br />
<div class="caption center"><br />
Hollow fiber bioreactor system<br />
</div><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-15"><br />
In Nov 2004, the estimated cost of dredging to remove the mercury contaminated mud in the lake was determined to be $451 million.<sup>[3]</sup> Currently the cost of our hollow fiber bioreactor system is about $560 with the cost being largely due to the reactor itself ($490) and the remainder of the cost was for the pump and filters. <br />
<br> <br><br />
However, it should be noted that the scale of the hollow fiber bioreactor system is much smaller as its volume is about 1L. Hence, the hollow fiber bioreactor would have to be scaled up significantly (by about 10<sup>11</sup> times!) in order to have any impact. To give a better idea of the scale, if the lake were the size of an Olympic swimming pool, the volume of the hollow fiber bioreactor would be equivalent to a drop of water. While we would need to scale up the volume of our hollow fiber bioreactor, it should also be noted that by placing the bioreactors in series, better mercury sequestration is achieved.<sup>[1]</sup> <br />
Therefore, even though it has been shown that the hollow fiber bioreactor is successful on a pilot scale, more tests would be required to determine if it is as effective on a larger scale. As there are several variables that might change e.g. flow rates and membrane area, the performance of the engineered <i>E. coli</i> might not simply scale up as expected. Nevertheless, given the environmental costs associated with existing remediation methods such as dredging, it is important to look into how biological systems are able to complement these solutions and solve the problem of mercury contamination in an effective, safe and cost efficient manner. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Chen, S., Kim, E., Shuler, M., & Wilson, D. (1998). Hg2+ Removal by Genetically Engineered Escherichia coli in a Hollow Fiber Bioreactor. Biotechnology Progress, 667-671</li><br />
<li>Moriarty, Rick. "Discovering What Lies at the Bottom of Onondaga Lake." Syracuse.com. Syracuse.com, n.d. Web. 24 Sept. 2014.</li><br />
<li>Collin, Glen. "Onondaga Lake Dredging Begins for Season; Could End a Year Early (video)." Syracuse.com. N.p., 7 Apr. 2014. Web. 11 Aug. 2014.</li><br />
</ol><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/modelingTeam:Cornell/project/modeling2014-10-18T01:23:48Z<p>E.Holmes: </p>
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<div class="row"><br />
<div class="col-md-8 col-xs-15"><br />
<h1>Effectiveness and economic feasibility of our hollow fiber bioreactor system</h1><br />
To determine the effectiveness and economic feasibility of our hollow fiber bioreactor with <br />
<i> E. coli</i> which has been engineered to express a mercury transport system and metallothionein, we modeled its impact when applied to a current real situation, mercury pollution in Onondaga Lake, Syracuse, NY.<br />
<br> <br><br />
It has been shown that similar hollow fiber bioreactors are able to reduce the concentration of mercury from 2mg/L to about 5 µg/L.<sup>[1]</sup> This corresponds to a promising 99.8% reduction in mercury levels. Furthermore as discussed in our case study, Onondaga Lake has a capacity of 35 billion gallons and about 165,000 lbs of mercury has been dumped into the lake over the years.[2] This corresponds to an approximate mercury concentration of 0.56 mg/L. Thus, the mercury concentration is Onondaga Lake is within the limits that the engineered <i> E. coli</i> is able to sequester. <br />
</div><br />
<div class="col-md-4 col-xs-5"><br />
<div class="thumbnail"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e0/Cornell_Onondaga_Lake_Park.jpg" alt="Onondaga Lake Park: Syracuse, NY"><br />
<div class="caption center"><br />
Onondaga Lake Park: Syracuse, NY<br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-4 col-xs-5"><br />
<div class="thumbnail" style="margin-top:0;"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/CORNELL2014_OveralBox.JPG" alt="Hollow fiber bioreactor system"> <br />
<div class="caption center"><br />
Hollow fiber bioreactor system<br />
</div><br />
</div><br />
</div><br />
<div class="col-md-8 col-xs-15"><br />
In Nov 2004, the estimated cost of dredging to remove the mercury contaminated mud in the lake was determined to be $451 million.<sup>[3]</sup> Currently the cost of our hollow fiber bioreactor system is about $560 with the cost being largely due to the reactor itself ($490) and the remainder of the cost was for the pump and filters. <br />
<br> <br><br />
However, it should be noted that the scale of the hollow fiber bioreactor system is much smaller as its volume is about 1L. Hence, the hollow fiber bioreactor would have to be scaled up significantly (by about <sup>10</sup> times!) in order to have any impact. To give a better idea of the scale, if the lake were the size of an Olympic swimming pool, the volume of the hollow fiber bioreactor would be equivalent to a drop of water. While we would need to scale up the volume of our hollow fiber bioreactor, it should also be noted that by placing the bioreactors in series, better mercury sequestration is achieved.<sup>[1]</sup> <br />
Therefore, even though it has been shown that the hollow fiber bioreactor is successful on a pilot scale, more tests would be required to determine if it is as effective on a larger scale. As there are several variables that might change e.g. flow rates and membrane area, the performance of the engineered <i>E. coli</i> might not simply scale up as expected. Nevertheless, given the environmental costs associated with existing remediation methods such as dredging, it is important to look into how biological systems are able to complement these solutions and solve the problem of mercury contamination in an effective, safe and cost efficient manner. <br />
</div><br />
</div><br />
<div class="row"><br />
<div class="col-md-12 col-xs-18"><br />
<h1 style="margin-bottom: 0px">References</h1><br />
<hr><br />
<ol><br />
<li>Chen, S., Kim, E., Shuler, M., & Wilson, D. (1998). Hg2+ Removal by Genetically Engineered Escherichia coli in a Hollow Fiber Bioreactor. Biotechnology Progress, 667-671</li><br />
<li>Moriarty, Rick. "Discovering What Lies at the Bottom of Onondaga Lake." Syracuse.com. Syracuse.com, n.d. Web. 24 Sept. 2014.</li><br />
<li>Collin, Glen. "Onondaga Lake Dredging Begins for Season; Could End a Year Early (video)." Syracuse.com. N.p., 7 Apr. 2014. Web. 11 Aug. 2014.</li><br />
</ol><br />
</div><br />
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</html></div>E.Holmeshttp://2014.igem.org/Team:Cornell/project/hprac/environTeam:Cornell/project/hprac/environ2014-10-18T01:16:19Z<p>E.Holmes: </p>
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<h1>Environmental Water Samples</h1><br />
We collaborated with seven other iGEM teams to test the quality of environmental water across the United States. RHIT, IvyTech, Northwestern, BYU, UCSC, UCSF-UCB, and Yale iGEM teams were kind enough to send us 50 mL water samples from creeks, rivers, and lakes in their local areas. We tested these samples for heavy metal contaminants to gauge the extent of heavy metal pollution across the United States. We also provided each of our collaborating teams a water quality report to inform them of the quality of their local water and, if warranted, to guide future remediation efforts. A compilation of these water quality reports can be found <a href="https://static.igem.org/mediawiki/2014/8/8a/CornelliGEM_2014waterqualityreport.pdf">here</a>. Of the 9 total samples that we tested for heavy metals, 4 contained a measurable amount of nickel, four contained a measurable amount of lead, and three contained a measurable amount of mercury. These metals tended to be present together, as three samples contained all three contaminants and one sample contained two. While these concentrations were not incredibly high, measured concentrations of mercury and lead both exceeded the maximum allowable levels for drinking water. What this data suggests is that even in the United States where strict regulations are put on drinking water quality and waste disposal, heavy metal pollution is still a widespread problem that needs to be addressed.<br />
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<a class="thumbnail" data-toggle="lightbox" a href="https://static.igem.org/mediawiki/2014/e/ea/Cornell_Water_Sample_Collaboration.png"> <br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Cornell_Water_Sample_Collaboration.png"><br />
<div class="caption center">Locations of water samples tested</div><br />
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<img src="https://static.igem.org/mediawiki/2014/5/54/Cornell_Environmental_contaminants.png"><br />
<div class="caption center">Water samples received from collaborating teams</div><br />
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<h1>Construct Design</h1><br />
To allow for the transport and sequestration of mercury ions into <i>E. coli</i> cells, genes that encode for the cellular production of heavy metal transport proteins and metallothioneins have been added to the pSB1C3 high copy bacterial plasmid. The mercury transport system is composed of <i>merT</i> and <i>merP</i>, genes originally found in <i>Pseudomonas aeruginosa</i>. <i>merP</i> is a periplasmic mercury ion scavenging protein. <i>merT</i> is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm.The <i>merT</i> and <i>merP</i> coupled transport system has been used in previous studies to develop luminescence based biosensors for the detection of mercury in the surroundings of bacterial cells. <br />
<br><br><br />
Our BioBrick BBa_K1460004 is composed of the Anderson promoter followed by a ribosomal binding site, <i>merT</i>, <i>merP</i>, and a terminator. The constitutive Anderson promoter allows for the constant expression of metal uptake proteins within our engineered <i>E. coli</i>. The BioBrick <a href="http://parts.igem.org/Part:BBa_K1460007">BBa_K1460007</a> is a composite of parts <a href="http://parts.igem.org/Part:BBa_K1460004">BBa_K1460004</a> and <a href="http://parts.igem.org/Part:BBa_K1460001">BBa_K1460001</a>, and it contains the mercury transport proteins (along with promoter, ribosomal binding site, and terminator) upstream of the GST-<i>crs5</i> metallothionein gene in pSB1C3. By coupling the <i>merT</i> and <i>merP</i> system with metallothionein, we hope to develop an effective biological system for our cells to uptake mercury ions and bind intracellularly to metallothioneins. <br />
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<img src="https://static.igem.org/mediawiki/2014/9/9b/Cornell_Mercury_crop.png"><br />
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<h3>BBa_K1460007</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>merT</i> and <i>merP</i> should be transporting more mercury ions past the cell wall. This would lead to increased mercury sensitivity. Additionally, cells expressing <i>merT</i> and <i>merP</i> as well as metallothionein should have increased tolerance to mercury due to the presence of metallothionein. To test for mercury sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460004 in the cm<sup>r</sup> plasmid pSB1C3 were grown for a 24 hour period in LB with .5 uM Hg (a mercury concentration we found to have moderate toxicity to wild type BL21 cells. To test for increased metal tolerance, we grew <i>E.coli</i> BL21 and engineered BL21 with parts BBa_K1460001 (GST-YMT in pSB1C3) and BBa_K1460004 (<i>merT/merP</i> in pUC57) in 5 uM Hg (a mercury concentration we found to be very toxic to wild type BL21 cells). <br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/8/82/Cornell_merTandmerP_growth.png"><br />
<img src="https://static.igem.org/mediawiki/2014/8/82/Cornell_merTandmerP_growth.png"><br />
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<a class="thumbnail" data-toggle="lightbox" href="https://static.igem.org/mediawiki/2014/1/1c/Cornell_merandmet_growth.png"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1c/Cornell_merandmet_growth.png"> <br />
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What we observe in both cases is what we expect. We see that BL21 engineered with BBa_K1460004 has impaired growth when compared to wild type BL21 (figure 1). This suggests that, in fact, BBa_K1460004 acts as expected and engineered cells successfully transport more mercury ions past the membrane than wild type cells. When BL21 engineered with both <i>merT/merP</i> and GST-<i>crs5</i> are grown in a highly toxic concentration of mercury we see significant growth when in wild type BL21 we do not (figure 2). This suggests that these cells are successfully expressing metallothionein and that this metallothionein is providing the cells with an inherent resistance to mercury toxicity. <br />
<br><br><br />
Part BBa_K1460004 in pUC57 was co-transformed with part BBa_K1460001 (GST-<i>crs5</i>) in pSB1C3 and selected for with both ampicillin and chloramphenicol to effectively create the mercury sequestration part BBa_K1460007. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460004 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Hg for a final mercury concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for mercury 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.<br />
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There was no statistically significant difference between BL21 wild type and BL21 engineered to express <i>merT/merP</i> and GST-<i>crs5</i> in final culture concentration of mercury or mercury sequestered per OD. This result prevents us from definitively confirming that the engineered bacteria are capable of sequestering mercury. The mercury concentrations used in this test were much higher than was shown in growth experiments to completely prevent growth of BL21, so it is likely that cells were quickly killed once metal was added, possibly confounding results. To verify this construct is successful in removing mercury from water, we must repeat these experiments using lower concentrations of Hg. We were not able to complete these experiments, however, as the limit of detection of the ICP-AES used to test these metal concentrations is above the uM range necessary to conduct these experiments. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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<ol><br />
<li>Lund, P., & Brown, N. (1987). Role of the <i>merT</i> and <i>merP</i> gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene, 207-214.</li><br />
<li>Omura, T., Kiyono, M., & Pan-Hou, H. (2004). Development of a Specific and Sensitive Bacteria Sensor for Detection of Mercury at Picomolar Levels in Environment. Journal of Health Science, 379-383.</li><br />
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<h1>Future Work</h1><br />
In the future, we hope to continue working with the <i>merT</i>, <i>merP</i>, <i>CBP4</i>, and <i>nixA</i> heavy metal transport genes by incorporating them upstream of the GST and <i>crs5</i> metallothionein sequestration genes. Once each heavy metal transport gene is combined with the metallothionein gene, we can transform the high copy bacterial plasmid into <i>E. coli</i>. We will then be able to conduct a series of growth assays between our engineered bacteria and <i>E. coli</i> in the presence of heavy metal contaminated water. <br />
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We also hope to continue working on synthesizing a reporter system. In order to detect the saturation of metallothionein sequestering cultures, we plan on using <i>amilCP</i> behind the nickel/cobalt activated promoter P<i>rcn</i> and the mercury activated promoter P<i>merT</i>. It would be useful to place <i>amilCP</i> behind a lead activated promoter. This system should be incorporated into the BioBrick backbone and transformed into <i>E. coli</i> reporter cultures. These would theoretically be placed into a second hollow fiber reactor that would be connected downstream to the transporter-metallothionein hollow fiber reactor. Effluent water carrying unsequestered metal ions would induce the reporter culture to express <i>amilCP</i>, producing a gradient of blue. We can then test water samples with different heavy metal concentrations to correlate effluent levels against the cultures’ color gradient.<br />
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<h1>Future Work</h1><br />
In the future, we hope to continue working with the <i>merT</i>, <i>merP</i>, <i>CBP4</i>, and <i>nixA</i> heavy metal transport genes by incorporating them upstream of the <i>GST</i> and <i>crs5</i> metallothionein sequestration genes. Once each heavy metal transport gene is combined with the metallothionein gene, we can transform the high copy bacterial plasmid into <i>E. coli</i>. We will then be able to conduct a series of growth assays between our engineered bacteria and <i>E. coli</i> in the presence of heavy metal contaminated water. <br />
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We also hope to continue working on synthesizing a reporter system. In order to detect the saturation of metallothionein sequestering cultures, we plan on using <i>amilCP</i> behind the nickel/cobalt activated promoter P<i>rcn</i> and the mercury activated promoter P<i>merT</i>. It would be useful to place <i>amilCP</i> behind a lead activated promoter. This system should be incorporated into the BioBrick backbone and transformed into <i>E. coli</i> reporter cultures. These would theoretically be placed into a second hollow fiber reactor that would be connected downstream to the transporter-metallothionein hollow fiber reactor. Effluent water carrying unsequestered metal ions would induce the reporter culture to express <i>amilCP</i>, producing a gradient of blue. We can then test water samples with different heavy metal concentrations to correlate effluent levels against the cultures’ color gradient.<br />
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<h1>Construct Design</h1> <br />
In order to introduce heavy metal ions into our bacteria and allow the metallothionein proteins to bind and sequester these contaminants, we created BioBricks for the expression of heavy metal membrane transporters. The gene <i>cpb4</i> codes for a membrane transporter that has a high capacity for the uptake of lead as well as a reduced affinity for other heavy metals, notably cadmium and cobalt. This gene was originally isolated from a resistant strain of <i>Bacillus spp.</i> found in heavy metal contaminated soil in Korea, although our plasmids utilize the gene from the plant <i>Nicotiana tabacum</i>. It has been found in previous research that bacterial strains possessing this gene have the capacity to remove lead from water and soil and could be useful in bioremediation applications <sup>[1]</sup>.<br />
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Our first lead transporter construct <a href="http://parts.igem.org/Part:BBa_K1460005">(BBa_K1460005)</a> consists of the constitutive Anderson promoter and the <i>cbp4</i> gene for constitutive expression of the heavy metal membrane transporter and uptake of lead. The primary construct for lead sequestration <a href="http://parts.igem.org/Part:BBa_K1460008">(BBa_K1460008)</a> consists of this first lead transporter construct put upstream of our metallothionein construct with the <i>T7</i> promoter and <i>GST-crs5</i>. This construct allows for the constitutive expression of the lead transporter as well as the inducible expression of the metallothionein in <i>BL21</i> by arabinose activating the <i>araBAD</i> promoter and allowing expression of the highly active T7 polymerase. This allows for our bacterial strains to grow to stationary phase before being induced to produce metallothioneins and being used to sequester lead.<br />
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<h3>BBa_K1460008</h3><br />
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<h1>Results</h1><br />
Cells successfully expressing <i>cbp4</i> should be transporting more lead ions past the cell wall. This would lead to increased lead sensitivity. To test for lead sensitivity, <i>E.coli</i> BL21 and engineered BL21 with part BBa_K1460005 in the amp<sup>r</sup> plasmid pUC57 were grown for a 24 hour period in LB with 1 mM Pb.<br />
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What we see after 24 hours of growth is no significant difference in growth between the two strains (figure 1). However, what we consistently observed is that there is no inhibition of growth of BL21 at high concentrations of lead (figure 2). Even if <i>cpb4</i> is expressed and is actively transporting lead ions into cells, it is possible that the concentration of lead is still not high enough to be toxic to the organisms. We were, unfortunately, unable to test lead concentrations higher than those shown above because these working concentrations are approaching the maximum solubility of the lead nitrate that we were using for testing. <br />
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Part BBa_K1460005 in pUC57 was co-transformed with part <a href="https://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 lead sequestration part BBa_K1460008. To test for sequestration efficiency, both BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 were grown with LB + 0.1% Arabinose for 8 hours and then diluted in half with LB + 2 mM Pb for a final lead concentration of 1 mM. These cultures were grown for 8 more hours. The cells were then removed and supernatant was tested for lead 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. <br />
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The chart on the left shows the average final concentration of lead in the cultures. There was no statistically significant difference between BL21 and BL21 engineered with BBa_K1460001 and BBa_K1460005 (figure 3). 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. This data suggests that cells engineered with <i>cbp4</i> and GST-<i>crs5</i> are in fact able to remove lead ions from water, and to the best of our knowledge this is the <b>first</b> successful bacterial lead sequestration system involving transport proteins and metallothioneins. <br />
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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 as, <a href="https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein#MTresults">as we have shown</a>, cells expressing metallothionein have inhibited growth. <br />
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<h1 style="margin-bottom: 0px">References</h1><br />
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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><br />
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