eau d’e coliigem 2006: mit

meet the teamThe Undergrads:Kate Broadbent Biological Engineering

Andre Green Biology / Physics

Stephen Payne Biological Engineering

Veena Venkatachalam Chemistry / Physics

Boyuan Zhu Electrical Engineering and Computer Science

The Grad Students:Barry CantonAustin CheJason KellyReshma ShettySamantha Sutton

The Faculty:Drew EndyTom Knight

motivations

eau d’e coli

mit igem 2006

background

A. thaliana

JMTjasmine

O. basilicu

m

CCMTcinnamon

A. majus

BAMTfresh / floral

BSMTwintergreen

C. breweri S. cerevisia

e

ATF1banana

background

O. basilicu

m

CCMTcinnamon

A. majus

BAMTfresh / floral

BSMTwintergreen

C. breweri S. cerevisia

e

ATF1banana

background

A. majus

BAMTfresh / floral

BSMTwintergreen

C. breweri S. cerevisia

e

ATF1banana

background

BSMTwintergreen

C. breweri S. cerevisia

e

ATF1banana

timeline:initial planning

initial planning • scent production • self-generating scent • production regulation • eliminating indole

timeline:scent productioninitial planning • scent production • self-generating scent • production regulation •

eliminating indole

timeline:self-generating scent

initial planning • scent production • self-generating scent • production regulation • eliminating indole

timeline:production regulation

initial planning • scent production • self-generating scent • production regulation • eliminating indole

timeline:indole eliminationinitial planning • scent production • self-generating scent • production regulation •

eliminating indole

wintergreen generating device

device generates wintergreen

O

O

OH

methyl salicylateMW = 152no methyl

salicylate [no peak at 9.1 minutes]

E. coli E. coli + precursor (SA) + WGD

banana generating device

precursor biosynthesis

precursor biosynthesis

goal: the regulated system

growth dependent regulation

osmY: active in stationary phase & under high osmotic pressure conditions

osmY

osmY + inverter

regulation is growth dependent

0E+0

2E+4

4E+4

6E+4

8E+4

1E+5

1E+5

1E+5

2E+5

2E+5

2E+5

0.0 0.1 0.2 0.3 0.4 0.5

Absorbance 600nm (relative units)

GFP Signal (relative units)

BBa_J45995 (pOsmY-GFP)

BBa_J45017 (Negative control)

BBa_I7100 (Constitutive promoter-GFP)

regulation is growth dependent

regulated system: exponential

regulated system: stationary

regulated system

chassis (tnaA5-) does not produce indole

No indole [no peak at 10

minutes]

IndolePeak at 10 minutes

MW = 117

wild type E. coli chassis: tnaA5- E. coli

final system

smell test results

0

10

20

30

40

50

60

70

80

90

100

A (Mint) B (Stinky) C (Banana)

Percentage

Banana

Mint

Stinky

future applications

Improve the workplace environment for microbiologists working with E. coli

Port our system to bacterial species involved in bioremediation.

Implement our system in bacteria responsible for bad human odor in the mouth, armpits and feet.

Implement our system in yeast used in bread and beer production

conclusion

Designed, built, and tested a synthetic biological system over the course of summer

Engineered synthetic scent systems in E. coli

Regulated via transcription control devices

acknowledgmentsNatalia Dudareva, Purdue University: gifts of expression vectors encoding BAMT, SAMT and BSMT enzymes.

Eran Pichersky, University of Michigan: suggestion of eliminating indole from Escherichia coli to mitigate the natural "bad" smell.

Mary Berlyn, CGSC, The Coli Genetic Stock Center: Esherichia coli strain YYC912.

Brian Cook, MIT: valuable discussions.

Cornelia Reimmann, University of Lausanne: gift of an expression vector carrying the pchBA coding region.

Dieter Haas, University of Lausanne: gift of the Pseudomonas fluorescens strain CHA0.

Peter Bakker, Utrecht University, The Netherlands: gift of an expression vector carrying the pmsCEAB coding region, as well as, gift of the Pseudomonas fluorescens strain WCS374.

Herbert P. Schweizer, Colorado State University: gift of the pUCP22 Esherichia coli to Pseudomonas shuttle vector.

Pamela Silver, Harvard Medical School: gift of yeast BioBricks vector.

questions and answers

growth curves

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0 10000 20000 30000 40000 50000 60000 70000

Time (min)

Absorbance 600nm (relative units)

Bba_J45995 (pOsmY-GFP)

Bba_J45017 (Negative control)

BBa_I7100 (Constitutive promoter-GFP)