eau d’e coli igem 2006: mit. meet the team the undergrads: kate broadbent biological engineering...
TRANSCRIPT
![Page 1: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/1.jpg)
eau d’e coliigem 2006: mit
![Page 2: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/2.jpg)
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
![Page 3: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/3.jpg)
motivations
eau d’e coli
mit igem 2006
![Page 4: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/4.jpg)
background
A. thaliana
JMTjasmine
O. basilicu
m
CCMTcinnamon
A. majus
BAMTfresh / floral
BSMTwintergreen
C. breweri S. cerevisia
e
ATF1banana
![Page 5: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/5.jpg)
background
O. basilicu
m
CCMTcinnamon
A. majus
BAMTfresh / floral
BSMTwintergreen
C. breweri S. cerevisia
e
ATF1banana
![Page 6: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/6.jpg)
background
A. majus
BAMTfresh / floral
BSMTwintergreen
C. breweri S. cerevisia
e
ATF1banana
![Page 7: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/7.jpg)
background
BSMTwintergreen
C. breweri S. cerevisia
e
ATF1banana
![Page 8: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/8.jpg)
timeline:initial planning
initial planning • scent production • self-generating scent • production regulation • eliminating indole
![Page 9: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/9.jpg)
timeline:scent productioninitial planning • scent production • self-generating scent • production regulation •
eliminating indole
![Page 10: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/10.jpg)
timeline:self-generating scent
initial planning • scent production • self-generating scent • production regulation • eliminating indole
![Page 11: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/11.jpg)
timeline:production regulation
initial planning • scent production • self-generating scent • production regulation • eliminating indole
![Page 12: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/12.jpg)
timeline:indole eliminationinitial planning • scent production • self-generating scent • production regulation •
eliminating indole
![Page 13: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/13.jpg)
wintergreen generating device
![Page 14: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/14.jpg)
device generates wintergreen
O
O
OH
methyl salicylateMW = 152no methyl
salicylate [no peak at 9.1 minutes]
E. coli E. coli + precursor (SA) + WGD
![Page 15: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/15.jpg)
banana generating device
![Page 16: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/16.jpg)
precursor biosynthesis
![Page 17: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/17.jpg)
precursor biosynthesis
![Page 18: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/18.jpg)
goal: the regulated system
![Page 19: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/19.jpg)
growth dependent regulation
osmY: active in stationary phase & under high osmotic pressure conditions
osmY
osmY + inverter
![Page 20: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/20.jpg)
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)
![Page 21: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/21.jpg)
regulation is growth dependent
![Page 22: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/22.jpg)
regulated system: exponential
![Page 23: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/23.jpg)
regulated system: stationary
![Page 24: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/24.jpg)
regulated system
![Page 25: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/25.jpg)
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
![Page 26: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/26.jpg)
final system
![Page 27: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/27.jpg)
smell test results
0
10
20
30
40
50
60
70
80
90
100
A (Mint) B (Stinky) C (Banana)
Percentage
Banana
Mint
Stinky
![Page 28: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/28.jpg)
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
![Page 29: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/29.jpg)
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
![Page 30: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/30.jpg)
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.
![Page 31: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/31.jpg)
questions and answers
![Page 32: Eau d’e coli igem 2006: mit. meet the team The Undergrads: Kate Broadbent Biological Engineering Andre Green Biology / Physics Stephen Payne Biological](https://reader036.vdocument.in/reader036/viewer/2022081602/551af8e855034607418b457e/html5/thumbnails/32.jpg)
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)