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Purifying Arsenic Polluted Water with Engineered Yeast . Matthew Alpert Shailendra Singh Shen-Long Tsai Dr. Ashok Mulchandani Dr. Wilfred Chen. Arsenic Toxicity. Common as As(V) and As(III). Arsenic Toxicity. Common as As(V) and As(III)

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Purifying arsenic polluted water with engineered yeast l.jpg

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)


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Arsenic Toxicity

  • Common as As(V) and As(III)

  • 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)

  • 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


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Arsenic Toxicity

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

  • 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

  • Disrupting the endocrine system has a serious impact on:

    • Metabolism

    • Tissue function

    • Growth and development


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Hyperkeratosis

Arsenic Toxicity


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Hyperkeratosis

Hypertension:

Strokes

Heart failure

Arterial aneurysm

Arsenic Toxicity


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Hyperkeratosis

Hypertension:

Strokes

Heart failure

Arterial aneurysm

Cancer:

Skin cancer

Lung cancer

Kidney cancer

Bladder cancer

Arsenic Toxicity


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Arsenic Pollution

  • Herbicide

  • Insecticide

  • Pesticide


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Arsenic Pollution

  • Herbicide

  • Insecticide

  • Pesticide

    • Brain damage has been found in those working the sprayers


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Arsenic Pollution

  • Herbicide

  • Insecticide

  • Pesticide

    • Brain damage has been found in those working the sprayers

  • Kingicide


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Arsenic Pollution

  • Herbicide

  • Insecticide

  • Pesticide

    • Brain damage has been found in those working the sprayers

  • 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)


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Arsenic Pollution

  • 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)

    • Protects wood from various forms of decay

    • The arsenic acts as an insecticide

  • 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

    • Coal, gold, etc.

  • 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

    • Granites containing cooper and tin


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Arsenic Pollution

  • Arsenic is found in various geological formations

    • Granites containing cooper and tin

  • Natural wearing can lead to its release


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Arsenic Pollution

  • Arsenic is found in various geological formations

    • Granites containing cooper and tin

  • Natural wearing can lead to its release

  • 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 PollutionWorldwide

Source: World Bank


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Arsenic PollutionUnited States

  • For decades the regulatory limit for arsenic was set to 50 g/L (ppb)


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Arsenic PollutionUnited States

  • 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)


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Arsenic PollutionUnited States

  • 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)

  • 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 PollutionUnited States


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Cleanup

  • An inexpensive and efficient method for arsenic remediation is needed


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Cleanup

  • An inexpensive and efficient method for arsenic remediation is needed

  • Most existing methods are impractical


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Cleanup

  • An inexpensive and efficient method for arsenic remediation is needed

  • 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


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Yeast

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

  • 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

  • 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)

    • Reduced to As(III) and dealt with accordingly




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Cysteine

  • As(III) has an affinity for thiol groups


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Cysteine

  • As(III) has an affinity for thiol groups

  • 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

  • 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


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Bioremediation

  • Efficiency must be increased


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Bioremediation

  • Efficiency must be increased

  • Saccharomyces cerevisiae 15616 Acr3Δ


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Bioremediation

  • Efficiency must be increased

  • Saccharomyces cerevisiae 15616 Acr3Δ

    • The Acr3p membrane transporter is deleted

    • As(III) is prevented from reentering the water


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Bioremediation

  • Efficiency must be increased

  • Saccharomyces cerevisiae 15616 Acr3Δ

    • The Acr3p membrane transporter is deleted

    • As(III) is prevented from reentering the water

  • Arabidopsis thaliana phytochelatin synthase


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Bioremediation

  • Efficiency must be increased

  • Saccharomyces cerevisiae 15616 Acr3Δ

    • The Acr3p membrane transporter is deleted

    • As(III) is prevented from reentering the water

  • Arabidopsis thaliana phytochelatin synthase

    • Increase the quantity of available PCs for binding As(III)



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Bioremediation

  • Treponema denticola cysteine desulfhydrase


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Bioremediation

  • Treponema denticola cysteine desulfhydrase

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












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Current Progress

  • Dual plasmid system


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Current Progress

  • Dual plasmid system

    • Phytochelatin synthase

      Vector pYES2 (5857 bp)

      Uracil selection


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Current Progress

  • 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


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Current Work

  • Test for effectiveness

    • Phytochelatin levels


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Current Work

  • Test for effectiveness

    • Phytochelatin levels

    • Sulfide levels


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Current Work

  • Test for effectiveness

    • Phytochelatin levels

    • Sulfide levels

    • Arsenic accumulation


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Future Work

  • Experiment with over expression


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Future Work

  • Experiment with over expression

    • Phytochelatin levels

    • Sulfide levels

    • Acr2p levels


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Future Work

  • Experiment with over expression

    • Phytochelatin levels

    • Sulfide levels

    • Acr2p levels

  • Selection media


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Future Work

  • Experiment with over expression

    • Phytochelatin levels

    • Sulfide levels

    • Acr2p levels

  • Selection media

    • Plasmid loss


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Future Work

  • Experiment with over expression

    • Phytochelatin levels

    • Sulfide levels

    • Acr2p levels

  • Selection media

    • Plasmid loss

  • Cysteine levels during growth


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Acknowledgements

  • 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.

  • BRITE REU



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