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E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
} {1Sunday, November 1, 2009
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2Sunday, November 1, 2009
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E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
} {3Sunday, November 1, 2009
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Currently Detectable CompoundsMotivation
4Sunday, November 1, 2009
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0
OnOff
Binary
Towards Sensitive Analog DetectionMotivation
5Sunday, November 1, 2009
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0
OnOff
Binary
Towards Sensitive Analog DetectionMotivation
Analog, Threshold-Based
5Sunday, November 1, 2009
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Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {6Sunday, November 1, 2009
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Data & Results: Analog Promoters
Meet pBAD
5’...ATTTTTA...3’Wildtype
BBa_I13453
7Sunday, November 1, 2009
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Data & Results: Analog Promoters
Meet the pBAD Family
5’...ATTTTTA...3’Wildtype
BBa_I13453
8Sunday, November 1, 2009
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Data & Results: Analog Promoters
Meet the pBAD Family
5’...ATTTTTA...3’Wildtype
BBa_I13453
5’...AAGATAG...3’ StrongBBa_K206000
5’...TTTTTTA...3’Weak
BBa_K206001
8Sunday, November 1, 2009
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Data & Results: Analog Promoters
pBAD Promoter Test
5’...ATTTTTA...3’
5’...AAGATAG...3’
5’...TTTTTTA...3’
WildtypeBBa_I13453
StrongBBa_K206000
WeakBBa_K206001
9Sunday, November 1, 2009
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Data & Results: Analog Promoters
Qualitative Results
Weak
Wild Type
Strong
2.35 µM[Arabinose]
10Sunday, November 1, 2009
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Data & Results: Analog Promoters
Time Course of RFP ExpressionR
FP F
luor
esce
nce
(arb
itrar
y un
its)
Time (hours)
pBAD Strong (K206000)
pBAD Wild Type (I13453)
pBAD Weak (K206001)
Control
11Sunday, November 1, 2009
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Data & Results: Analog Promoters
Transfer Function of RFP Expression
Arabinose Concentration (µM)
RFP
Flu
ores
cenc
e (a
rbitr
ary
units
)
2.35 µM
pBAD Strong (K206000)
pBAD Wild Type (I13453)
pBAD Weak (K206001)
12Sunday, November 1, 2009
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Data & Results: Analog Promoters
Highly Specific pBADsR
FP F
luor
esce
nce
(arb
itrar
y un
its)
250
200
150
100
50
0xylose lyxose rhamnose arabinoseribose
pBAD Strong (K206000)
pBAD Wild Type (I13453)
pBAD Weak (K206001)
Control
13Sunday, November 1, 2009
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Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {14Sunday, November 1, 2009
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Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84715Sunday, November 1, 2009
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Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84715Sunday, November 1, 2009
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Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84716Sunday, November 1, 2009
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Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84717Sunday, November 1, 2009
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Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {18Sunday, November 1, 2009
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Color CrosstalkChallenge: The Traffic Light Switcher
19Sunday, November 1, 2009
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Color CrosstalkChallenge: The Traffic Light Switcher
+
19Sunday, November 1, 2009
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Color CrosstalkChallenge: The Traffic Light Switcher
+
19Sunday, November 1, 2009
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Color CrosstalkChallenge: The Traffic Light Switcher
+
+ +
19Sunday, November 1, 2009
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Color CrosstalkChallenge: The Traffic Light Switcher
+
+ +
19Sunday, November 1, 2009
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No Truly Modular Repression?
USTC iGEM 2007“Logic Promoters”
BBa_732205
A B
Challenge: The Traffic Light Switcher
20Sunday, November 1, 2009
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No Truly Modular Repression?
USTC iGEM 2007“Logic Promoters”
BBa_732205
A B
Challenge: The Traffic Light Switcher
Concentration-DependentRepression
20Sunday, November 1, 2009
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RNAi for prokaryotic chassis?
Exogenous RNA Introduced
Eukaryotic Processing(e.g. DICER, RISC)
Gene Repression(e.g. GFP knockdown)
Challenge: The Traffic Light Switcher
21Sunday, November 1, 2009
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RNAi for prokaryotic chassis?
Exogenous RNA Introduced
Eukaryotic Processing(e.g. DICER, RISC)
Gene Repression(e.g. GFP knockdown)
Not autonomous
Not chassis-independent
Challenge: The Traffic Light Switcher
22Sunday, November 1, 2009
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Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
23Sunday, November 1, 2009
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Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
24Sunday, November 1, 2009
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Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
24Sunday, November 1, 2009
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Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
25Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
We need a solution...
GFP Fluorescence
% o
f Max
imum
Arabinose +Arabinose -Positive Control
Negative Control
Incomplete Jammer
26Sunday, November 1, 2009
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Meet the Jammer v.2
Data & Results: The Traffic Light Switcher
27Sunday, November 1, 2009
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Meet the Jammer v.2
Data & Results: The Traffic Light Switcher
27Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
We found the solution
GFP Fluorescence
% o
f Max
imum
Arabinose +Arabinose -Positive Control
Negative Control
Complete Jammer
28Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
29Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
30Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
31Sunday, November 1, 2009
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Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
32Sunday, November 1, 2009
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Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {33Sunday, November 1, 2009
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The Traffic LightDesign
34Sunday, November 1, 2009
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The Traffic LightDesign
35Sunday, November 1, 2009
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The Traffic LightDesign
36Sunday, November 1, 2009
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The Traffic LightDesign
37Sunday, November 1, 2009
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The Traffic LightDesign
38Sunday, November 1, 2009
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The Traffic LightDesign
39Sunday, November 1, 2009
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The Traffic LightDesign
40Sunday, November 1, 2009
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The Traffic LightDesign
41Sunday, November 1, 2009
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The Traffic LightDesign
42Sunday, November 1, 2009
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The Traffic LightDesign
43Sunday, November 1, 2009
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The Traffic LightDesign
44Sunday, November 1, 2009
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The Traffic LightDesign
45Sunday, November 1, 2009
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The Traffic LightDesign
46Sunday, November 1, 2009
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The Traffic LightDesign
47Sunday, November 1, 2009
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The Traffic LightDesign
48Sunday, November 1, 2009
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The Traffic LightDesign
49Sunday, November 1, 2009
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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The Traffic LightDesign
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Our ContributionsConclusions
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Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
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Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
Jammer: Modular Repression Technology
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Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
Jammer: Modular Repression Technology
Traffic Light Biosensor using Threshold
Detection
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One more thing...
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Applications: The Next Big ThingsOne more thing...
}{
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Applications: The Next Big ThingsOne more thing...
Organ Rejection Biomarkers
MMP9
Off
PhysiologicalEffectors
Inhibitor Production}{
}{
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Applications: The Next Big ThingsOne more thing...
Organ Rejection Biomarkers
MMP9
Off
PhysiologicalEffectors
Inhibitor Production}{
}{Environmental Homeostasis
Inorganic Iron
Iron Sequestration
Off
Iron Release }{
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a BIG THANK YOU to...Acknowledgements
Teaching andLearningEnhancementFund
• Dept. of Chemical & Biological Engineering• Genome BC• Integrated DNA Technologies
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a BIG THANK YOU to...Acknowledgements
Dr. Eric T. LagallyFaculty Sponsor
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a BIG THANK YOU to...Acknowledgements
Dr. Eric T. LagallyFaculty Sponsor
Soon-to-be-Dr. Paul JaschkeGraduate Advisor
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a BIG THANK YOU to...Acknowledgements
Alex NgAmelia Hardjasa
Calvin ChanCharles Howes
Eric MaHank Yu
Heather KempthorneJanny Ke
Karen CheshireMark Ling
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E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
Questions
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Rationale for pBAD MutationsQ&A
Niland, Huhne, Muller-Hill (1996) J. Mol. Biol. 264(4:667-674)
AraC Binding Affinity to pBAD
Rate of Initiation of Transcription
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Lock and Key Activation SystemQ&A
Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.
+
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Lock and Key Activation SystemQ&A
Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.
+
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RNAi-like pathways in ProkaryotesQ&A
RNase III degrades dsRNA
Meng et al. (2008) Methods Enzymol. 447(119-129)
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