1. 2007 Brown iGEM Team 7 undergraduates 7 grad student advisors
2 Faculty advisors
9 faculty sponsors
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2. Brown iGEM
June Update
international genetically engineered machines competition
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3. What is iGEM?
Biology
Engineering
Standardization
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4. Science - Nuts and Bolts
Standardizing biology
Systematic engineering
Apply biological technology
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5. Previous Projects
Bacterial Photo Film - U. Texas (published in Nature)
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6. Previous Projects Sepsis Treatment - Slovenia
Banana/Wintergreen Smelling Cells - MIT
Arsenic Water Detection - Edinburgh
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7. Anderson, J. C. , Clarke, E. J. , Arkin, A. P. , & Voigt, C. A
Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2005) Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria, Journal of Molecular Biology
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8. Aerobic Conditions
Low Cell Density
>0.02% Arabinose
OFF
Hypoxia
High Cell Density
<0.02% Arabinose ON
Inv
induction
INVASION
Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2005) Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria, Journal of Molecular Biology
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9. by a team of undergraduates
Undergraduate Teams
+ Imagined
+ Designed
+ Implemented
by a team of undergraduates
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10. Progress Brown’s 2nd year UTRA grants
Lab space in Multi Disciplinary Lab
Equipment sponsorship
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11. Project 1: Lead Biosensor
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12. Why do we need this?
Public health concern: Lead in soil, paint, water, dust
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13. Why do we need this?
Public health concern: Lead in soil, paint, water, dust
Lead Poisoning is often caused by ingesting contaminated drinking water, or soil. It can cause neurological and gastrointestinal disorders, especially among children.
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14. Why do we need this?
Public health concern: Lead in soil, paint, water, dust
Lead Poisoning is often caused by ingesting contaminated drinking water, or soil. It can cause neurological and gastrointestinal disorders, especially among children.
The legal limit of lead in drinking water is 15 parts per billion.
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15. Why do we need this?
Current ways of testing for lead either require expensive chemical lab analysis or involve inaccurate home kits.
So why do we want a biosensor?
- Cheap
- Sensitive
- Quick
- Specific
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16. We have spliced together biobrick parts into plasmids in E. Coli
We have spliced together biobrick parts into plasmids in E. Coli. The bacteria express our genetic devices to create a lead detector.
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17. General Design Lead Detection Lead Detection Signal Amplification
Fluorescent Output
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18. 17/45

19. Part 2: Signal Amplification
2 System Components
Part 1: Lead Detection
Part 2: Signal Amplification
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20. Part 1: Lead Detection Lead Receptor Protein Lead Receptor Gene
Always On
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21. Activated Lead Receptor Protein
Part 1: Lead Detection
Lead
Activated Lead Receptor Protein
Lead Receptor Protein
Lead Receptor Gene
Always On
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22. Part 1: Lead Detection Lead Activated Lead Receptor Protein
Lead Receptor Gene
Always On
Signal Amplification promoter
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23. Part 1: Lead Detection
This is sensitive ONLY to lead; other metals will not activate it.
Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44:2715–2719
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Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44:2715–2719

24. General Design Lead Detection Lead Detection Signal Amplification
Fluorescent Output
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25. Part 2: Signal Amplification
Activated Lead Receptor Protein
Signal Amplification promoter
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26. Part 2: Signal Amplification
Message
Activated Lead Receptor Protein
Message Producer Gene
Signal Amplification promoter
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27. Part 2: Signal Amplification
Message Producer Gene
Signal (GFP)
Message
Activated Lead Receptor Protein
Message Producer Gene
Signal Amplification promoter
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28. Part 2: Signal Amplification
Message Producer Gene
Signal (GFP)
Message
Activated Lead Receptor Protein
Repeated activation of this promoter causes amplification of the signal.
Message Producer Gene
Signal Amplification promoter
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29. 3 System Components Lead Detection Signal Amplification
1. Detector Sequence
2. Leakiness Filter to Eliminate False Positives
Signal Amplification
3. Positive Feedback Loop for Amplification
We’ve added a “Leakiness Filter” as an additional component to the system. This gives our circuit one more level of complexity. However, the black box diagram is generally the same.
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30. NO LEAD Lead Activator Message Activator Always On
Transcription factors are constitutively made by the first promoter.
Message Maker
Filter Repressor
Lead Detector
Message Destroyer
Filter
Filter Eliminates Stray Message Molecules to Prevent False Feedback Loop Activation
These proteins are poised to activate the Lead Detector promoter and Message Receiver promoter upon addition of lead.
Stray Message Molecules Can Falsely Activate the Feedback Loop.
Message Maker
GFP Reporter
Message Receiver
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31. + Lead Activator Feedback Activator Always On Message Maker
Filter Repressor
Lead turns on Detector promoter
Lead Detector
Message Destroyer
Leakiness Filter promoter gets turned off
Filter +
Fluorescent Protein Output
Message Maker
GFP Reporter
Message Receiver
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32. Lead Activator
Feedback Activator
Always On
Message Maker
Filter Repressor 1
Lead Detector
Message Destroyer 2
Filter
Message Maker
GFP Reporter
Message Receiver 3
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33. PbrR
LuxR
pTet
LuxI
LacI 1
Pbr
aiiA 2
pLac
LuxI
GFP
pLux
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34. 33/45

35. How this project advances science:
Characterization of Existing Parts
Adding New Parts and Devices
Lead Promoter and Transcription Factor
Amplifier Device
Generalize to future biosensors
Arsenic, Cadmium, Mercury, Zinc
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36. Project 2:
THE TRI-STABLE SWITCH
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37. C A B What is Tri-stability?
A tri-stable switch has three distinct and inducible states C A B
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38. Achieving Tri-stability
Input A
Output A
State A
Input B
Output B
State B
Input C
Output C
State C
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39. The Architecture Arabinose RFP pBad/Ara tetR lacI IPTG CFP pLac araC
Tetracycline
YFP
pTet
lacI
araC
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40. Characterization Characterization is an essential aspect of iGEM
It is a step towards standardization - giving others all the details needed to use the part.
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41. Uses Differentiation of stem cells
Turn on/off three different proteins in cell
Cellular logic
Tissue Engineering
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42. Timeline Now: August: End of lab work November: Jamboree at MIT
Start cloning
PCR lead promoter
Clone tri-stable switch
Characterize parts
Test systems
Send back to the Registry
August: End of lab work
November: Jamboree at MIT
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43. Why Brown?
Innovators
Entrepreneurs
A great place for new ideas!
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44. The Future Educate others about iGEM and synthetic biology
Synthetic Biology Course Offering in Fall 07! Led by Prof. Gary Wessel
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45. Special Thanks To:
Office of the Dean of the College Office of the President The Atlantic Philanthropies The Center for Computational and Molecular Biology Department of Physics Engineering Department Department of Molecular Biology, Cell Biology, and Biochemistry Department of Molecular Pharmacology, Physiology, and Biotechnology The Multi Disciplinary Lab Pfizer Labnet Nanodrop
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46. Thank you for listening! Questions?
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