1. eau d’e coli igem 2006: mit

2. meet the team The Undergrads: The Grad Students: The Faculty:
Kate Broadbent
Biological Engineering
Andre Green
Biology / Physics
Stephen Payne
Veena Venkatachalam
Chemistry / Physics
Boyuan Zhu
Electrical Engineering and Computer Science
The Grad Students:
Barry Canton
Austin Che
Jason Kelly
Reshma Shetty
Samantha Sutton
The Faculty:
Drew Endy
Tom Knight
Shrink text

3. motivations eau d’e coli mit igem 2006
Hasn’t been explored much in synthetic bio
Put Cool, Regulated biosynthetic devices in e. coli for the registry
eau d’e coli
mit igem 2006

4. background A. thaliana O. basilicum A. majus C. breweri S. cerevisiae
Started out considering various diff scents; for various reasons, we didn’t do things.
evaluated several possibilities; chose one
JMT
jasmine
CCMT
cinnamon
BAMT
fresh / floral
BSMT
wintergreen
ATF1
banana

5. background O. basilicum A. majus C. breweri S. cerevisiae CCMT BAMT
Bold titles on top
Show five different pics of diff scents, to show
CCMT
cinnamon
BAMT
fresh / floral
BSMT
wintergreen
ATF1
banana

6. background A. majus C. breweri S. cerevisiae BAMT BSMT ATF1
Bold titles on top
Show five different pics of diff scents, to show
BAMT
fresh / floral
BSMT
wintergreen
ATF1
banana

7. background C. breweri S. cerevisiae BSMT ATF1 banana wintergreen
Bold titles on top
Show five different pics of diff scents, to show
BSMT
wintergreen
ATF1
banana

8. timeline: initial planning
initial planning • scent production • self-generating scent • production regulation • eliminating indole

9. timeline: scent production
initial planning • scent production • self-generating scent • production regulation • eliminating indole
Mention WGD = wintergreen generating device

10. timeline: self-generating scent
initial planning • scent production • self-generating scent • production regulation • eliminating indole

11. timeline: production regulation
initial planning • scent production • self-generating scent • production regulation • eliminating indole
Short: Growth dependent regulation

12. timeline: indole elimination
initial planning • scent production • self-generating scent • production regulation • eliminating indole

13. wintergreen generating device
Once again….redundant titles?

14. device generates wintergreen
methyl salicylate
MW = 152
no methyl salicylate [no peak at 9.1 minutes]
E. coli
E. coli + precursor (SA) + WGD
Fix titles

15. banana generating device

16. precursor biosynthesis
Fix arrows

17. precursor biosynthesis
Mention: we put these devices together w/ WGD and BGD, and found that they expressed the scent without exogenously adding precursor

18. goal: the regulated system
Pcred out of e. coli; added to registry

19. growth dependent regulation
osmY: active in stationary phase & under high osmotic pressure conditions
Reason for regulation: smell when cultures are done
osm Y
osmY + inverter

20. regulation is growth dependent
Tested it using fluorescent protein techniques

21. regulation is growth dependent
Tested it using fluorescent protein techniques

22. regulated system: exponential
Arrow going to chorismate from inside the cell; methyl salicylate and isoamyl acetate out of the cell (no arrows to banana and mint smell)
Corner box: graph

23. regulated system: stationary
IT WORKED
Same phrase: “we designed this part, constructed it, tested it, proved that it worked, and added it to the registry”

24. regulated system
Haven’t verified if it’s correct; but we’ve tested all the individual parts

25. chassis (tnaA5-) does not produce indole
No indole
[no peak at 10 minutes]
Indole
Peak at 10 minutes
MW = 117
wild type E. coli
chassis: tnaA5- E. coli
Mention yale genomic stock center strain #
Rename: wild type e. coli and “chassis: [name of strain] e. coli”

26. final system Change title of cell to indole-deficient
Get rid of title of indole knockout chassis in other slides.

27. smell test results

28. 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

29. 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

30. acknowledgments
Natalia 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.
Get rid of depts.

31. questions and answers

32. growth curves