e. coli traffic light biosensor2009.igem.org/files/presentation/british_columbia.pdf · 2009. 11....
TRANSCRIPT
E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
} {1Sunday, November 1, 2009
2Sunday, November 1, 2009
E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
} {3Sunday, November 1, 2009
Currently Detectable CompoundsMotivation
4Sunday, November 1, 2009
0
OnOff
Binary
Towards Sensitive Analog DetectionMotivation
5Sunday, November 1, 2009
0
OnOff
Binary
Towards Sensitive Analog DetectionMotivation
Analog, Threshold-Based
5Sunday, November 1, 2009
Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {6Sunday, November 1, 2009
Data & Results: Analog Promoters
Meet pBAD
5’...ATTTTTA...3’Wildtype
BBa_I13453
7Sunday, November 1, 2009
Data & Results: Analog Promoters
Meet the pBAD Family
5’...ATTTTTA...3’Wildtype
BBa_I13453
8Sunday, November 1, 2009
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
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
Data & Results: Analog Promoters
Qualitative Results
Weak
Wild Type
Strong
2.35 µM[Arabinose]
10Sunday, November 1, 2009
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
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
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
Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {14Sunday, November 1, 2009
Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84715Sunday, November 1, 2009
Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84715Sunday, November 1, 2009
Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84716Sunday, November 1, 2009
Data & Results: Lock and Key System
Rapid Response Activation
Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84717Sunday, November 1, 2009
Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {18Sunday, November 1, 2009
Color CrosstalkChallenge: The Traffic Light Switcher
19Sunday, November 1, 2009
Color CrosstalkChallenge: The Traffic Light Switcher
+
19Sunday, November 1, 2009
Color CrosstalkChallenge: The Traffic Light Switcher
+
19Sunday, November 1, 2009
Color CrosstalkChallenge: The Traffic Light Switcher
+
+ +
19Sunday, November 1, 2009
Color CrosstalkChallenge: The Traffic Light Switcher
+
+ +
19Sunday, November 1, 2009
No Truly Modular Repression?
USTC iGEM 2007“Logic Promoters”
BBa_732205
A B
Challenge: The Traffic Light Switcher
20Sunday, November 1, 2009
No Truly Modular Repression?
USTC iGEM 2007“Logic Promoters”
BBa_732205
A B
Challenge: The Traffic Light Switcher
Concentration-DependentRepression
20Sunday, November 1, 2009
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
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
Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
23Sunday, November 1, 2009
Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
24Sunday, November 1, 2009
Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
24Sunday, November 1, 2009
Meet the Jammer v.1
Data & Results: The Traffic Light Switcher
25Sunday, November 1, 2009
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
Meet the Jammer v.2
Data & Results: The Traffic Light Switcher
27Sunday, November 1, 2009
Meet the Jammer v.2
Data & Results: The Traffic Light Switcher
27Sunday, November 1, 2009
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
Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
29Sunday, November 1, 2009
Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
30Sunday, November 1, 2009
Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
31Sunday, November 1, 2009
Data & Results: The Traffic Light Switcher
Jammer: Any Input, Any Output
32Sunday, November 1, 2009
Parts of a Traffic LightDesign
Lock & KeyActivation
Traffic LightSwitching
DetectionUsing Sensitive
Promoters
} {33Sunday, November 1, 2009
The Traffic LightDesign
34Sunday, November 1, 2009
The Traffic LightDesign
35Sunday, November 1, 2009
The Traffic LightDesign
36Sunday, November 1, 2009
The Traffic LightDesign
37Sunday, November 1, 2009
The Traffic LightDesign
38Sunday, November 1, 2009
The Traffic LightDesign
39Sunday, November 1, 2009
The Traffic LightDesign
40Sunday, November 1, 2009
The Traffic LightDesign
41Sunday, November 1, 2009
The Traffic LightDesign
42Sunday, November 1, 2009
The Traffic LightDesign
43Sunday, November 1, 2009
The Traffic LightDesign
44Sunday, November 1, 2009
The Traffic LightDesign
45Sunday, November 1, 2009
The Traffic LightDesign
46Sunday, November 1, 2009
The Traffic LightDesign
47Sunday, November 1, 2009
The Traffic LightDesign
48Sunday, November 1, 2009
The Traffic LightDesign
49Sunday, November 1, 2009
The Traffic LightDesign
50Sunday, November 1, 2009
The Traffic LightDesign
51Sunday, November 1, 2009
The Traffic LightDesign
52Sunday, November 1, 2009
The Traffic LightDesign
53Sunday, November 1, 2009
The Traffic LightDesign
54Sunday, November 1, 2009
The Traffic LightDesign
55Sunday, November 1, 2009
The Traffic LightDesign
56Sunday, November 1, 2009
The Traffic LightDesign
57Sunday, November 1, 2009
Our ContributionsConclusions
58Sunday, November 1, 2009
Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
58Sunday, November 1, 2009
Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
Jammer: Modular Repression Technology
58Sunday, November 1, 2009
Our ContributionsConclusions
Family of Concentration
Sensitive Promoters
Jammer: Modular Repression Technology
Traffic Light Biosensor using Threshold
Detection
58Sunday, November 1, 2009
One more thing...
59Sunday, November 1, 2009
Applications: The Next Big ThingsOne more thing...
}{
60Sunday, November 1, 2009
Applications: The Next Big ThingsOne more thing...
Organ Rejection Biomarkers
MMP9
Off
PhysiologicalEffectors
Inhibitor Production}{
}{
60Sunday, November 1, 2009
Applications: The Next Big ThingsOne more thing...
Organ Rejection Biomarkers
MMP9
Off
PhysiologicalEffectors
Inhibitor Production}{
}{Environmental Homeostasis
Inorganic Iron
Iron Sequestration
Off
Iron Release }{
60Sunday, November 1, 2009
a BIG THANK YOU to...Acknowledgements
Teaching andLearningEnhancementFund
• Dept. of Chemical & Biological Engineering• Genome BC• Integrated DNA Technologies
61Sunday, November 1, 2009
a BIG THANK YOU to...Acknowledgements
Dr. Eric T. LagallyFaculty Sponsor
62Sunday, November 1, 2009
a BIG THANK YOU to...Acknowledgements
Dr. Eric T. LagallyFaculty Sponsor
Soon-to-be-Dr. Paul JaschkeGraduate Advisor
62Sunday, November 1, 2009
a BIG THANK YOU to...Acknowledgements
Alex NgAmelia Hardjasa
Calvin ChanCharles Howes
Eric MaHank Yu
Heather KempthorneJanny Ke
Karen CheshireMark Ling
63Sunday, November 1, 2009
E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team
Questions
64Sunday, November 1, 2009
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
65Sunday, November 1, 2009
Lock and Key Activation SystemQ&A
Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.
+
66Sunday, November 1, 2009
Lock and Key Activation SystemQ&A
Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.
+
66Sunday, November 1, 2009
RNAi-like pathways in ProkaryotesQ&A
RNase III degrades dsRNA
Meng et al. (2008) Methods Enzymol. 447(119-129)
67Sunday, November 1, 2009