Joe Bozzay Maria Fini Brandon Freshcorn …2009.igem.org/files/presentation/Virginia.pdfMaria Fini...

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/