2007 brown igem team
DESCRIPTION
2007 Brown iGEM Team. 7 undergraduates 7 grad student advisors 2 Faculty advisors 9 faculty sponsors. 1/45. Brown iGEM. international genetically engineered machines competition. June Update. 2/45. What is iGEM?. Biology Engineering Standardization. 3/45. Science - Nuts and Bolts. - PowerPoint PPT PresentationTRANSCRIPT
2007 Brown iGEM Team
•7 undergraduates
•7 grad student advisors
•2 Faculty advisors
•9 faculty sponsors
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Brown iGEMinternational genetically engineered machines competition
June Update
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What is iGEM?
• Biology
• Engineering
• Standardization
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Science - Nuts and Bolts
•Standardizing biology
•Systematic engineering
•Apply biological technology
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Previous Projects
•Bacterial Photo Film - U. Texas (published in Nature)
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Previous Projects
•Sepsis Treatment - Slovenia
•Banana/Wintergreen Smelling Cells - MIT
•Arsenic Water Detection - Edinburgh
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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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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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Undergraduate Teams
+ Imagined+ Designed
+ Implemented
by a team of undergraduates
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Progress
•Brown’s 2nd year
•UTRA grants
•Lab space in Multi Disciplinary Lab
•Equipment sponsorship
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Project 1: Lead Biosensor
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Why do we need this?Public health concern: Lead in soil, paint, water, dust
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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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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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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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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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Lead Detection
Signal Amplification
Fluorescent Output
Lead
General Design
Lead Detection
Signal Amplification
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2 System Components
Part 1: Lead Detection
Part 2: Signal Amplification
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Always On
Lead Receptor
Gene
Lead Receptor Protein
Part 1: Lead Detection
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Always On
Lead Receptor
Gene
Lead Receptor Protein
Lead
Activated Lead
Receptor Protein
Part 1: Lead Detection
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Activated Lead
Receptor Protein
Always On
Lead Receptor
Gene
Lead Receptor Protein
Lead
Activated Lead
Receptor Protein
Signal Amplification
promoter
Part 1: Lead Detection
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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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Lead Detection
Signal Amplification
Fluorescent Output
Lead
General Design
Lead Detection
Signal Amplification
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Activated Lead
Receptor Protein
Signal Amplification
promoter
Part 2: Signal Amplification
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Activated Lead
Receptor Protein
Signal Amplification
promoter
Part 2: Signal Amplification
Message Producer
Gene
Message
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Activated Lead
Receptor Protein
Signal Amplification
promoter
Part 2: Signal Amplification
Message Producer
Gene
MessageMessage Producer
Gene
Signal (GFP)
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Activated Lead
Receptor Protein
Signal Amplification
promoter
Part 2: Signal Amplification
Message Producer
Gene
MessageMessage Producer
Gene
Signal (GFP)
Repeated activation of this promoter causes amplification of the signal.
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3 System Components
Lead Detection
Signal Amplification
1. Detector Sequence
2. Leakiness Filter to Eliminate False Positives
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. 28/45
Always On
Lead Activator
Message Activator
Lead Detector
Message Maker
Filter Repressor
FilterMessage Destroyer
Message Receiver
Message Maker
GFP Reporter
Stray Message Molecules Can Falsely Activate the Feedback
Loop.
Filter Eliminates Stray Message Molecules to
Prevent False Feedback Loop
Activation
NO LEADTranscription factors
are constitutively made by the first promoter.
These proteins are poised to activate the
Lead Detector promoter and Message Receiver promoter upon addition
of lead.29/45
Always On
Lead Activator
Feedback Activator
Lead Detector
Message Maker
Filter Repressor
FilterMessage Destroyer
Message Receiver
Message Maker
GFP Reporter
+Fluorescent
Protein Output
Lead turns on Detector promoter
Leakiness Filter promoter gets turned off
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Always On
Lead Activator
Feedback Activator
Lead Detector
Message Maker
Filter Repressor
FilterMessage Destroyer
Message Receiver
Message Maker
GFP Reporter
1
2
3 31/45
pTetPbrR LuxR
PbrLuxI LacI
pLacaiiA
pLuxLuxI GFP
1
2
3 32/45
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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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THE TRI-STABLESWITCH
Project 2:
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What is Tri-stability?• A tri-stable
switch has three distinct and inducible states
ABC
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Achieving Tri-stability
State A
State C
State B
Output A
Output B
Output C
Input A
Input CInput B
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pBad/Ara tetR
tetR
lacI
lacI
pLac araC
araCpTet
RFP
CFP
YFP
The ArchitectureArabinose
IPTG
Tetracycline
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Characterization• Characterization is an essential aspect of iGEM
• It is a step towards standardization - giving others all the details needed to use the part. 39/45
Uses
• Differentiation of stem cells
• Turn on/off three different proteins in cell
• Cellular logic• Tissue Engineering
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Timeline• Now:
– 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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Why Brown?
•Innovators
•Entrepreneurs
•A great place for new ideas!
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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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Office of the Dean of the CollegeOffice of the President
The Atlantic PhilanthropiesThe Center for Computational and Molecular Biology
Department of PhysicsEngineering Department
Department of Molecular Biology, Cell Biology, and Biochemistry
Department of Molecular Pharmacology, Physiology, and Biotechnology
The Multi Disciplinary LabPfizerLabnet
Nanodrop
Special Thanks To:
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Thank you for listening!
Questions?45/45