joe bozzay maria fini brandon freshcorn …2009.igem.org/files/presentation/virginia.pdfmaria fini...
TRANSCRIPT
Joe Bozzay
Maria FiniMaria Fini
Brandon Freshcorn
Rohini Manaktala
Dan Tarjan
Thaddeus Webb
Arsenic has significant toxicity
• Mechanisms
– As(III) binds thiol groups
• inhibits TCA cycle, leads to decreased cellular • inhibits TCA cycle, leads to decreased cellular
ATP
– As(V) resembles inorganic phosphate
• substitutes for phosphate in respiration, forms
ADP-arsenate instead of ATP
Biosequestration using E. coli
Arsenic Sponge
Sequestration
Absorb and Sequester
E. coli Arsenic pathway
Efflux
∆ArsB prevents As(III) efflux
Toxic
Metallothioneins
• Cysteine rich peptides
• Bind heavy metals via thiol group• Bind heavy metals via thiol group
• Naturally produced in some plants
• Reduce heavy metal toxicity
Phytochelatin synthase catalyses
formation of phytochelatin
Glutathione
(exists in E. coli)
GSH PCS PC
Phytochelatin
synthase
Phytochelatin
(metallothioneine)
Phytochelatin binds As(III)
ABP – Arsenic Binding Peptide
• Synthetic metallothionein
• Shown to bind Cadmium and Mercury
• Alternative to exogenous protein
expression
ABP binds As(III)
Parts
BBa_K231000BBa_K231001
Arsenic Binding Peptide Phytochelatin synthase
As(III) and As(V) Flux in E. coli
Understand, Predict, Design
Simulation for WT E. coli
∆ArsB Model predicts lower
extracellular [As(III)]
∆ArsB exhibits
normal growth
K12- AsV
K12- AsIII
∆ArsB - AsV
∆ArsB - AsIII
∆ArsB exhibits limited
growth in 2mM Arsenic
K12
∆ArsB
∆ArsB PCS induced cells show similar growth to controls 0.1mM AsV
∆ArsB ABP induced cells show similar growth to controls 0.1 mM AsV
∆ArsB PCS reduces arsenic in solution
1.00E-04
1.20E-04
1.40E-04
Ex
tra
cell
ula
r [A
rse
nic
] M
AsV Induced AsV Noninduced AsIII Induced AsIII Noninduced
0.00E+00
2.00E-05
4.00E-05
6.00E-05
8.00E-05
0 100 200 300 400 500 600 700 800
Ex
tra
cell
ula
r [A
rse
nic
] M
Time (min)
Results confirm model predictions
∆ArsB PCS+
∆ArsB PCS-
Human Practices• Synthetic Biology needs to maintain a positive
image and engage the media
• We actively pursued coverage of our work
Synthetic Biologist’s Guide to Speaking
with the Press
http://2009.igem.org/Team:Virginia
Sequestration
Ethics
Without the generous support of the
following organizations our
research would not be possible
We thank our invaluable advisors
Prof Kwon – Chemical Engineering
Prof Fernandez – Chemical Engineering
Prof Papin – Biomedical EngineeringThe University of Virginia Engineering
Foundation
The School of Engineering and Applied
Sciences
The College and Graduate School of Arts &
Sciences
The Office of the Vice President for Research
The School of Medicine
The Department of Biology
The Department of Chemical Engineering
DNA 2.0
Prof Papin – Biomedical Engineering
Prof Kozminski – Biology
Prof Timko – Biology
Kay Christopher
Kim Yasutis
Matt Oberhardt
The Virginia Commonwealth team
Arsenic Arsenic
Sponge
Existing treatment methods are too
expensive
• Reversal Osmosis System (cost: $800-$1,500)
– Small scale, 2-3 gallons/day
• Anionic Exchange System (cost: $2,000-$3,000 per • Anionic Exchange System (cost: $2,000-$3,000 per
household)
– Non-specific, other factors can compete to bind
resin
• Iron Oxide Filters (cost: $89.95 per cartridge)
HPLC Calibration
As(V)
As(III)
Set of Model ODEs Describing As(III)
and As(V) Flux in E. Coli
Model Sensitivity AnalysisC.V.=0.1
Model Sensitivity Analysis
Citations• Contamination of drinking-water by arsenic in Bangladesh: a public health emergency. Smith
et al. 2000. Bulletin WHO. 2000
• Leverone, J. Development of New Technologies for Removal of Arsenic from Water Supplies
Through Biomimicry of Natural Systems.
<http://edcsarasotacountry.com/documents/Arsenic%20Speciation%20and%20Toxicity%20R
eport.pdf>.
• Mandal, S., Mondal, K., Dey, S., Pati, B. A rapid colony screening method for the detection of
arsenate-reducing bacteria. Indian J. Microbiol. 47, 167-169 (2007).
• Meng, Y., Liu, Z. and Rosen, B.P. As(III) and Sb(III) uptake and efflux in Escherichia coli J. Biol.
Chem. 279, 18334-18341 (2004).
• Methallothionein. Wikipedia. <http://en.wikipedia.org/wiki/Metallothionein>.• Methallothionein. Wikipedia. <http://en.wikipedia.org/wiki/Metallothionein>.
• Pazirandeh, M., Wells, B., Ryan, R. Development of Bacterium-Based Heavy Metal
Biosorbents: Enhanced Uptake of Cadmium and Mercury by Excherichia coli Expressing a
Metal Binding Motif. Appl Environ Microbiol. 64, 4068-4072 (1998).
• Qin, J., Rosen, B., Zhang, Y., Wang, G., Franke, S., Rensing, C. Arsenic detoxification and
evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine
methyltransferase. PNAS. 108, 2075-2080 (2006).
• Rosen, B., Liu, Z. Transport pathways for arsenic and selenium: A minireview. Environ Intl. 35,
512-515 (2009).
• Lung X-ray - Wikimedia Commons.
• Clean hands - http://www.flickr.com/photos/aussiegall/