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Purifying Arsenic Polluted Water with Engineered Yeast Matthew Alpert Shailendra Singh Shen-Long Tsai Dr. Ashok Mulchandani Dr. Wilfred Chen

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Arsenic Toxicity Common as As(V) and As(III) Common as As(V) and As(III)

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Arsenic Toxicity Common as As(V) and As(III) Common as As(V) and As(III) As(V) can be substituted for phosphate in the citric acid cycle, interfering with: As(V) can be substituted for phosphate in the citric acid cycle, interfering with:

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Arsenic Toxicity Common as As(V) and As(III) Common as As(V) and As(III) As(V) can be substituted for phosphate in the citric acid cycle, interfering with: As(V) can be substituted for phosphate in the citric acid cycle, interfering with: The reduction of NAD + ATP synthesis Mitochondrial respiration

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Arsenic Toxicity As(III) acts as an endocrine disruptor by binding to hormone receptors As(III) acts as an endocrine disruptor by binding to hormone receptors

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Arsenic Toxicity As(III) acts as an endocrine disruptor by binding to hormone receptors As(III) acts as an endocrine disruptor by binding to hormone receptors Disrupting the endocrine system has a serious impact on: Disrupting the endocrine system has a serious impact on:

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Arsenic Toxicity As(III) acts as an endocrine disruptor by binding to hormone receptors As(III) acts as an endocrine disruptor by binding to hormone receptors Disrupting the endocrine system has a serious impact on: Disrupting the endocrine system has a serious impact on: Metabolism Tissue function Growth and development

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Arsenic Toxicity Hyperkeratosis Hyperkeratosis

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Arsenic Toxicity Hyperkeratosis Hyperkeratosis Hypertension: Hypertension: Strokes Heart failure Arterial aneurysm

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Arsenic Toxicity Hyperkeratosis Hyperkeratosis Hypertension: Hypertension: Strokes Heart failure Arterial aneurysm Cancer: Cancer: Skin cancer Lung cancer Kidney cancer Bladder cancer

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Arsenic Pollution Herbicide Herbicide Insecticide Insecticide Pesticide Pesticide

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Arsenic Pollution Herbicide Herbicide Insecticide Insecticide Pesticide Pesticide Brain damage has been found in those working the sprayers

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Arsenic Pollution Herbicide Herbicide Insecticide Insecticide Pesticide Pesticide Brain damage has been found in those working the sprayers Kingicide Kingicide

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Arsenic Pollution Herbicide Herbicide Insecticide Insecticide Pesticide Pesticide Brain damage has been found in those working the sprayers Kingicide Kingicide King George III of Great Britain Francesco I de' Medici, Grand Duke of Tuscany

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Arsenic Pollution Chromated copper arsenate (CCA) (Tanalith brand) Chromated copper arsenate (CCA) (Tanalith brand)

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Arsenic Pollution Chromated copper arsenate (CCA) (Tanalith brand) Chromated copper arsenate (CCA) (Tanalith brand) Protects wood from various forms of decay The arsenic acts as an insecticide

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Arsenic Pollution Chromated copper arsenate (CCA) (Tanalith brand) Chromated copper arsenate (CCA) (Tanalith brand) Protects wood from various forms of decay The arsenic acts as an insecticide Depending on application and environment, the amount of chemical leaching varies Depending on application and environment, the amount of chemical leaching varies

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Arsenic Pollution Arsenic is a waste product of some mining and smelting activities Arsenic is a waste product of some mining and smelting activities Coal, gold, etc. Poor handing leads to various forms of pollution, including arsenic in groundwater Poor handing leads to various forms of pollution, including arsenic in groundwater

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Arsenic Pollution Arsenic is found in various geological formations Arsenic is found in various geological formations Granites containing cooper and tin

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Arsenic Pollution Arsenic is found in various geological formations Arsenic is found in various geological formations Granites containing cooper and tin Natural wearing can lead to its release Natural wearing can lead to its release

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Arsenic Pollution Arsenic is found in various geological formations Arsenic is found in various geological formations Granites containing cooper and tin Natural wearing can lead to its release Natural wearing can lead to its release Wells may tap into polluted ground water Wells may tap into polluted ground water Wells are commonly used as a source of microbiologically safe drinking water without much though to possible chemical dangers

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Arsenic Pollution Worldwide Source: World Bank

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Arsenic Pollution United States For decades the regulatory limit for arsenic was set to 50 g/L (ppb) For decades the regulatory limit for arsenic was set to 50 g/L (ppb)

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Arsenic Pollution United States For decades the regulatory limit for arsenic was set to 50 g/L (ppb) For decades the regulatory limit for arsenic was set to 50 g/L (ppb) Recently the U.S. Environmental Protection Agency lowered the limit to only 10 g/L (ppb) Recently the U.S. Environmental Protection Agency lowered the limit to only 10 g/L (ppb)

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Arsenic Pollution United States For decades the regulatory limit for arsenic was set to 50 g/L (ppb) For decades the regulatory limit for arsenic was set to 50 g/L (ppb) Recently the U.S. Environmental Protection Agency lowered the limit to only 10 g/L (ppb) Recently the U.S. Environmental Protection Agency lowered the limit to only 10 g/L (ppb) Many sites which previously had safe levels of arsenic are now over the limit by as much as five times Many sites which previously had safe levels of arsenic are now over the limit by as much as five times

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Arsenic Pollution United States

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Cleanup An inexpensive and efficient method for arsenic remediation is needed An inexpensive and efficient method for arsenic remediation is needed

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Cleanup Most existing methods are impractical Most existing methods are impractical

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Cleanup An inexpensive and efficient method for arsenic remediation is needed An inexpensive and efficient method for arsenic remediation is needed Most existing methods are impractical Most existing methods are impractical They lack specificity Are small scale Require alteration of water chemistry Fail to remove trace quantities Or are completely ineffective against As(III) which is uncharged at natural pH

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Yeast Yeast has defense mechanisms to protect itself from heavy metals and metalloids Yeast has defense mechanisms to protect itself from heavy metals and metalloids

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Yeast As(III) As(III) Transported back out of the yeast cells Bound by glutathione and stored in vacuoles Bound by phytochelatins and sulfides forming various complexes

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Yeast Yeast has defense mechanisms to protect itself from heavy metals and metalloids Yeast has defense mechanisms to protect itself from heavy metals and metalloids As(III) As(III) Transported back out of the yeast cells Bound by glutathione and stored in vacuoles Bound by phytochelatins and sulfides forming various complexes As(V) As(V) Reduced to As(III) and dealt with accordingly

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Phytochelatin

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Cysteine

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Cysteine As(III) has an affinity for thiol groups As(III) has an affinity for thiol groups

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Cysteine This is why it binds to proteins, wreaking havoc on various organisms This is why it binds to proteins, wreaking havoc on various organisms

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Cysteine As(III) has an affinity for thiol groups As(III) has an affinity for thiol groups This is why it binds to proteins, wreaking havoc on various organisms This is why it binds to proteins, wreaking havoc on various organisms This is also how it binds to PC and is made harmless within the Yeast This is also how it binds to PC and is made harmless within the Yeast

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Bioremediation Efficiency must be increased Efficiency must be increased

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Bioremediation Saccharomyces cerevisiae 15616 Acr3 Saccharomyces cerevisiae 15616 Acr3

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Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3 Saccharomyces cerevisiae 15616 Acr3 The Acr3p membrane transporter is deleted As(III) is prevented from reentering the water

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Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3 Saccharomyces cerevisiae 15616 Acr3 The Acr3p membrane transporter is deleted As(III) is prevented from reentering the water Arabidopsis thaliana phytochelatin synthase Arabidopsis thaliana phytochelatin synthase

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Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3 Saccharomyces cerevisiae 15616 Acr3 The Acr3p membrane transporter is deleted As(III) is prevented from reentering the water Arabidopsis thaliana phytochelatin synthase Arabidopsis thaliana phytochelatin synthase Increase the quantity of available PCs for binding As(III)

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Bioremediation MicroorganismYeast E. coli ExpressAtPCSSpPCS Removal Efficiency LowHigh PC Level HighLow Sulfide Content LowHigh

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Bioremediation Treponema denticola cysteine desulfhydrase Treponema denticola cysteine desulfhydrase

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Bioremediation Available cysteine are stripped of their thiol groups, increasing the availability of free sulfides for binding As(III)

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Complexes

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Current Progress Dual plasmid system Dual plasmid system

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Current Progress Dual plasmid system Dual plasmid system Phytochelatin synthase Vector pYES2 (5857 bp) Uracil selection

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Current Progress Dual plasmid system Dual plasmid system Phytochelatin synthase Vector pYES2 (5857 bp) Uracil selection Cysteine desulfhydrase Vector YEplac181 (5741 bp) Leucine selection

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Current Work Test for effectiveness Test for effectiveness

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Current Work Test for effectiveness Test for effectiveness Phytochelatin levels

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Current Work Test for effectiveness Test for effectiveness Phytochelatin levels Sulfide levels

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Current Work Test for effectiveness Test for effectiveness Phytochelatin levels Sulfide levels Arsenic accumulation

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Future Work Experiment with over expression Experiment with over expression

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Future Work Experiment with over expression Experiment with over expression Phytochelatin levels Sulfide levels Acr2p levels

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Future Work Experiment with over expression Experiment with over expression Phytochelatin levels Sulfide levels Acr2p levels Selection media Selection media

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Future Work Experiment with over expression Experiment with over expression Phytochelatin levels Sulfide levels Acr2p levels Selection media Selection media Plasmid loss

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Future Work Experiment with over expression Experiment with over expression Phytochelatin levels Sulfide levels Acr2p levels Selection media Selection media Plasmid loss Cysteine levels during growth Cysteine levels during growth

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Acknowledgements This work was supported by grants from NSF (BES0422791 and BES0329482). This work was supported by grants from NSF (BES0422791 and BES0329482). Acknowledgements are due for Dr. Rea and Dr. Keasling for providing pYES3- AtPCS::FLAG and the cysteine desulfhydrase gene respectively. Acknowledgements are due for Dr. Rea and Dr. Keasling for providing pYES3- AtPCS::FLAG and the cysteine desulfhydrase gene respectively. BRITE REU BRITE REU

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Any questions?

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