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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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Presentation on theme: "Purifying Arsenic Polluted Water with Engineered Yeast Matthew Alpert Shailendra Singh Shen-Long Tsai Dr. Ashok Mulchandani Dr. Wilfred Chen."— Presentation transcript:

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

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

3 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:

4 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

5 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

6 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:

7 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

8 Arsenic Toxicity Hyperkeratosis Hyperkeratosis

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

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

11 Arsenic Pollution Herbicide Herbicide Insecticide Insecticide Pesticide Pesticide

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

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

14 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

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

16 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

17 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

18 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

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

20 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

21 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

22 Arsenic Pollution Worldwide Source: World Bank

23 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)

24 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)

25 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

26 Arsenic Pollution United States

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

28 Cleanup Most existing methods are impractical Most existing methods are impractical

29 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

30 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

31 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

32 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

33 Phytochelatin

34 Cysteine

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

36 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

37 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

38 Bioremediation Efficiency must be increased Efficiency must be increased

39 Bioremediation Saccharomyces cerevisiae 15616 Acr3Δ Saccharomyces cerevisiae 15616 Acr3Δ

40 Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3Δ Saccharomyces cerevisiae 15616 Acr3Δ –The Acr3p membrane transporter is deleted –As(III) is prevented from reentering the water

41 Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3Δ Saccharomyces cerevisiae 15616 Acr3Δ –The Acr3p membrane transporter is deleted –As(III) is prevented from reentering the water Arabidopsis thaliana phytochelatin synthase Arabidopsis thaliana phytochelatin synthase

42 Bioremediation Efficiency must be increased Efficiency must be increased Saccharomyces cerevisiae 15616 Acr3Δ Saccharomyces cerevisiae 15616 Acr3Δ –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)

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

44 Bioremediation Treponema denticola cysteine desulfhydrase Treponema denticola cysteine desulfhydrase

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

46 Complexes

47 Complexes

48 Complexes

49 Complexes

50 Complexes

51 Complexes

52 Complexes

53 Complexes

54 Complexes

55 Complexes

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80 Current Progress Dual plasmid system Dual plasmid system

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

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

83 Current Work Test for effectiveness Test for effectiveness

84 Current Work Test for effectiveness Test for effectiveness –Phytochelatin levels

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

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

87 Future Work Experiment with over expression Experiment with over expression

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

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

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

91 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

92 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

93 Any questions?

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