Brown iGEMinternational genetically engineered machines competition
August Update
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Brown iGEM 2007
• Lead Sensor
• Tristable Switch
• iGEM Jamboree on November 4th at MIT
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Lead Sensor
Introduction
Speaker: Deepa Galaiya
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Lead Detection
Signal Amplification
Fluorescent Output
Lead
General Design
Lead Detection
Signal Amplification
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Lead Sensor
Lead Detection
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PbrR691 Coding Region
PbrA Coding Region
Non-Coding Region
In Ralstonia metallidurans:
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PbrR691 Coding Region
PbrA Coding Region
Non-Coding Region
RBS
RBS
Lead Promoter
PbrR691 Promoter
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PbrR691 Coding Region
PbrA Coding Region
Non-Coding Region
3 versions
6 versions (Neils method)
3 versions
1 version
2 versions
15 total
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Planned Ligations
- All 15 parts into BioBrick plasmid- pTet to PbrR691 alone- All promoters and PbrR691
combinations to LuxI- All promoters and PbrR691
combinations to GFP- pTet-PbrR691 to promoters-LuxI- pTet-PbrR691 to promoters-GFP
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Completed Ligations
- All 15 parts into BioBrick plasmid
- pTet to PbrR691 alone (done today!)
- All promoters and PbrR691 combinations to LuxI
- All promoters and PbrR691 combinations to GFP
- pTet-PbrR691 to promoters-LuxI
- pTet-PbrR691 to promoters-GFP11/55
Results
• 15 parts into Biobrick gelled and sequenced
• PbrR691/promoter combinations in presence of lead nitrate give no GFP production and no AHL production compared to control.
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Discussion & Future Plans
PbrR691 most likely not expressed in combination parts. (Transcription factors needed?).
Promoter not abandoned yet!
Constitutive expression under pTet possible. Neils’ group was able to overexpress protein in E Coli under
IPTG control.
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Lead Sensor
The Amplifier
Speaker: Jeff Hofmann
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T9002
J37015 (The Amplifier)
Differences: Positive Feedback Loop Stronger Ribosome Binding Site
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How did we measure this?
GFP fluorescence – average control GFP fluorescence Cell Density
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Expected: Amplifier produces more GFP than T9002
Results: T9002 produces far more GFP than Amplifier(G
FP –
Con
trol
) /
Cell
Den
sity
Time (hours)
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Expected: Direct relationship between AHL input and GFP output
Results: Indirect relationship between AHL input and GFP output
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More AHL = Less GFP!
J37015 GFP output at 5 Hours
0
1000
2000
3000
4000
5000
6000
0 0.1 nM 1 nM 10 nM 100 nM 1 uM 10 uM 100 uM 1 mM
[AHL]
(GFP - control) / Cell Density
5 Hours
J37015 GFP output at 5 Hours
0
1000
2000
3000
4000
5000
6000
0 0.1 nM 1 nM 10 nM 100 nM 1 uM 10 uM 100 uM 1 mM
[AHL]
(GFP - control) / Cell Density
5 Hours
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Why does this happen? Possible wrong promoter GFP is further away from promoter in J37015
T9002
J37015 (The Amplifier)
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Why does this happen? Possible wrong promoter GFP is further away from promoter in J37015
T9002
J37015 (The Amplifier)
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Lead Sensor
Sequencing
Speaker: Rohan Maddamsetti
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Sequencing
• Importance of Sequencing
• Sequencing the Amplifier
• Sequencing the Lead Parts
• Where do we go from here?
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Tri-Stable Switch
Speaker: Kyle Schutter
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A
B C
The Tristable Switch
A switch with three distinctand inducible states.
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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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The Switch
pBADpBAD TetRTetRLacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
The Architecture as planned
C
A
B
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The Switch
pBADpBAD TetRTetRLacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
The Architecture as planned
L-arabinose
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The Switch
pBADpBAD TetRTetRLacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
The Architecture as planned
IPTG
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The Switch
pBADpBAD TetRTetRLacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
The Architecture as planned
anhydrotetracyclineanhydrotetracycline
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• Parts in the registry only allow two stable states and a third inducible state
• pBAD promoter is attached to gene
pBADpBAD TetRTetR LacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
pCpCAraCAraC
Architecture ReDesigned
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pBADpBAD TetRTetR LacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
pCpCAraCAraC
Architecture ReDesignedL-arabinose
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pBADpBAD TetRTetR LacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
pCpCAraCAraC
Architecture ReDesignedL-arabinose
But there is a lot of araC in the system33/55
pBADpBAD TetRTetR LacILacI
LacILacI
TetRTetRAraCAraC
AraCAraC
pLacpLac
pTetpTet
pCpCAraCAraC
Architecture ReDesigned
The system will fall into whichever of the
other two states is stronger.34/55
Characterization
• Registry not as well characterized/reliable as expected
– Rumor that araC gene has promoter region inside it
– All repressors LVA tagged: fast degradation leads to poor repression
– AraC connected to pBAD promoter
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iGEM Characterization Protocol
• Starting to formulate Characterization protocol for Promoters and Repressors
– Promoters: relative, how “on” or “off”
– Repressors: cooperativity, binding constant
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Tristable Switch
Creating the Parts
Speaker: Adam Emrich
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3 Major Steps to Create Parts
• Transformation of DNA, to make more DNA
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The 3 Major Steps
Transformation
Restriction
Ligation
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Purposes of each Step
Transformation: Increases the amount of DNA.
Extraction: Removes DNA from cells.
Restriction: Cuts DNA, to prepare it for Ligation.
Ligation: Attaches cut DNA, to create new genetic parts.
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Step 1: TransformationPurpose:
To create more DNA.
Method:
1. Insert DNA into specially prepared Competent Cells.
2. Plate out cells, allow to grow overnight.
3. Create overnight culture.
4. Extract DNA. 41/55
Step 2: RestrictionPurpose:
To cut DNA from Step 1, in preparation for Step 3: Ligation.
Method:
1. Insert DNA into a vial.
2. Add buffer and restriction enzymes, incubate 4-6 hours.
3. Heat inactivate enzymes. 42/55
Step 3: Ligation
Purpose:
To attach DNA from Restriction step together, resulting in new Genetic Parts.
Method:
???
We do not have an exact method for this yet.
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Protocol Determination
Transformation: ~Early July
Competent Cell Preparation: ~Early August
Ligation: ?
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Step 3: Ligation ProtocolOur ligation success rate is about 10%.
We are currently running experiments to determine a protocol that works at a higher success rate.
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Plan for the Future1. Establish a working protocol for Ligations
2. Work on project during semester
3. Assemble a Bi-Stable Switch by the Jamboree
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The Next Step
• Preparing for November 4th, MIT
• Continue progress
• Build
• Characterize
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Next Year
• Master protocols earlier
• Transformation to Ligation
• Problem-solving skills
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New Technologies
• Automated Assembly
• Synthesize all DNA
• New Equipment - Plate Reader
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Student Led
• iGEM Student Group (Fall ‘07)
• 2008 Applications judged by 2007 Team
• Be Selective
• Goal: 50 Applications
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Sponsors
Thank you
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John Cumbers
Alex BrodskyTayhas Palmore
Gary Wessel
Multidisciplinary Lab(Kathy, Shirley, Faye, Phil, Sarah)
CCMBMCB
MPPB Department of Physics Division of Engineering
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Brown iGEMinternational genetically engineered machines competition
August Update
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