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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

Purifying Arsenic Polluted Water with Engineered Yeast

Matthew Alpert

Shailendra Singh

Shen-Long Tsai

Dr. Ashok Mulchandani

Dr. Wilfred Chen

arsenic toxicity
Arsenic Toxicity
  • Common as As(V) and As(III)
arsenic toxicity3
Arsenic Toxicity
  • Common as As(V) and As(III)
  • As(V) can be substituted for phosphate in the citric acid cycle, interfering with:
arsenic toxicity4
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
arsenic toxicity5
Arsenic Toxicity
  • As(III) acts as an endocrine disruptor by binding to hormone receptors
arsenic toxicity6
Arsenic Toxicity
  • As(III) acts as an endocrine disruptor by binding to hormone receptors
  • Disrupting the endocrine system has a serious impact on:
arsenic toxicity7
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
arsenic toxicity9
Hyperkeratosis

Hypertension:

Strokes

Heart failure

Arterial aneurysm

Arsenic Toxicity
arsenic toxicity10
Hyperkeratosis

Hypertension:

Strokes

Heart failure

Arterial aneurysm

Cancer:

Skin cancer

Lung cancer

Kidney cancer

Bladder cancer

Arsenic Toxicity
arsenic pollution
Arsenic Pollution
  • Herbicide
  • Insecticide
  • Pesticide
arsenic pollution12
Arsenic Pollution
  • Herbicide
  • Insecticide
  • Pesticide
    • Brain damage has been found in those working the sprayers
arsenic pollution13
Arsenic Pollution
  • Herbicide
  • Insecticide
  • Pesticide
    • Brain damage has been found in those working the sprayers
  • Kingicide
arsenic pollution14
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
arsenic pollution15
Arsenic Pollution
  • Chromated copper arsenate (CCA) (Tanalith brand)
arsenic pollution16
Arsenic Pollution
  • Chromated copper arsenate (CCA) (Tanalith brand)
    • Protects wood from various forms of decay
    • The arsenic acts as an insecticide
arsenic pollution17
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
arsenic pollution18
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
arsenic pollution19
Arsenic Pollution
  • Arsenic is found in various geological formations
    • Granites containing cooper and tin
arsenic pollution20
Arsenic Pollution
  • Arsenic is found in various geological formations
    • Granites containing cooper and tin
  • Natural wearing can lead to its release
arsenic pollution21
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
arsenic pollution worldwide
Arsenic PollutionWorldwide

Source: World Bank

arsenic pollution united states
Arsenic PollutionUnited States
  • For decades the regulatory limit for arsenic was set to 50 g/L (ppb)
arsenic pollution united states24
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)
arsenic pollution united states25
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
cleanup
Cleanup
  • An inexpensive and efficient method for arsenic remediation is needed
cleanup28
Cleanup
  • An inexpensive and efficient method for arsenic remediation is needed
  • Most existing methods are impractical
cleanup29
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
yeast
Yeast
  • Yeast has defense mechanisms to protect itself from heavy metals and metalloids
yeast31
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
yeast32
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
cysteine35
Cysteine
  • As(III) has an affinity for thiol groups
cysteine36
Cysteine
  • As(III) has an affinity for thiol groups
  • This is why it binds to proteins, wreaking havoc on various organisms
cysteine37
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
bioremediation
Bioremediation
  • Efficiency must be increased
bioremediation39
Bioremediation
  • Efficiency must be increased
  • Saccharomyces cerevisiae 15616 Acr3Δ
bioremediation40
Bioremediation
  • Efficiency must be increased
  • Saccharomyces cerevisiae 15616 Acr3Δ
    • The Acr3p membrane transporter is deleted
    • As(III) is prevented from reentering the water
bioremediation41
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
bioremediation42
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)
bioremediation44
Bioremediation
  • Treponema denticola cysteine desulfhydrase
bioremediation45
Bioremediation
  • Treponema denticola cysteine desulfhydrase
    • Available cysteine are stripped of their thiol groups, increasing the availability of free sulfides for binding As(III)
current progress
Current Progress
  • Dual plasmid system
current progress81
Current Progress
  • Dual plasmid system
    • Phytochelatin synthase

Vector pYES2 (5857 bp)

Uracil selection

current progress82
Current Progress
  • Dual plasmid system
    • Phytochelatin synthase

Vector pYES2 (5857 bp)

Uracil selection

    • Cysteine desulfhydrase

Vector YEplac181 (5741 bp)

Leucine selection

current work
Current Work
  • Test for effectiveness
current work84
Current Work
  • Test for effectiveness
    • Phytochelatin levels
current work85
Current Work
  • Test for effectiveness
    • Phytochelatin levels
    • Sulfide levels
current work86
Current Work
  • Test for effectiveness
    • Phytochelatin levels
    • Sulfide levels
    • Arsenic accumulation
future work
Future Work
  • Experiment with over expression
future work88
Future Work
  • Experiment with over expression
    • Phytochelatin levels
    • Sulfide levels
    • Acr2p levels
future work89
Future Work
  • Experiment with over expression
    • Phytochelatin levels
    • Sulfide levels
    • Acr2p levels
  • Selection media
future work90
Future Work
  • Experiment with over expression
    • Phytochelatin levels
    • Sulfide levels
    • Acr2p levels
  • Selection media
    • Plasmid loss
future work91
Future Work
  • Experiment with over expression
    • Phytochelatin levels
    • Sulfide levels
    • Acr2p levels
  • Selection media
    • Plasmid loss
  • Cysteine levels during growth
acknowledgements
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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