E. coli Traffic Light Biosensor2009.igem.org/files/presentation/British_Columbia.pdf · 2009. 11. 16. · E. coli Traffic Light Biosensor British Columbia 2009 iGEM Team Questions

E. coli Traffic Light BiosensorBritish Columbia 2009 iGEM Team

} {1Sunday, November 1, 2009

2Sunday, November 1, 2009



Currently Detectable CompoundsMotivation


0

OnOff

Binary

Towards Sensitive Analog DetectionMotivation


0

OnOff

Binary

Towards Sensitive Analog DetectionMotivation

Analog, Threshold-Based


Parts of a Traffic LightDesign

Lock & KeyActivation

Traffic LightSwitching

DetectionUsing Sensitive

Promoters


Data & Results: Analog Promoters

Meet pBAD

5’...ATTTTTA...3’Wildtype

BBa_I13453



Meet the pBAD Family


BBa_I13453



Meet the pBAD Family


BBa_I13453

5’...AAGATAG...3’ StrongBBa_K206000

5’...TTTTTTA...3’Weak

BBa_K206001



pBAD Promoter Test

5’...ATTTTTA...3’

5’...AAGATAG...3’

5’...TTTTTTA...3’

WildtypeBBa_I13453

StrongBBa_K206000

WeakBBa_K206001



Qualitative Results

Weak

Wild Type

Strong

2.35 µM[Arabinose]



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



Transfer Function of RFP Expression

Arabinose Concentration (µM)

RFP

Flu

ores

cenc

e (a

rbitr

ary

units

)

2.35 µM



pBAD Weak (K206001)



Highly Specific pBADsR

FP F

luor

esce

nce

(arb

itrar

y un

its)

250

200

150

100

50

0xylose lyxose rhamnose arabinoseribose



pBAD Weak (K206001)

Control






Promoters


Data & Results: Lock and Key System

Rapid Response Activation

Isaacs et. al. Nat. Biotech. 2004: 22(7) pp. 841-84715Sunday, November 1, 2009














Promoters


Color CrosstalkChallenge: The Traffic Light Switcher



+



+



+

+ +



+

+ +


No Truly Modular Repression?

USTC iGEM 2007“Logic Promoters”

BBa_732205

A B

Challenge: The Traffic Light Switcher


No Truly Modular Repression?

USTC iGEM 2007“Logic Promoters”

BBa_732205

A B


Concentration-DependentRepression


RNAi for prokaryotic chassis?

Exogenous RNA Introduced

Eukaryotic Processing(e.g. DICER, RISC)

Gene Repression(e.g. GFP knockdown)



RNAi for prokaryotic chassis?

Exogenous RNA Introduced

Eukaryotic Processing(e.g. DICER, RISC)

Gene Repression(e.g. GFP knockdown)

Not autonomous

Not chassis-independent



Meet the Jammer v.1

Data & Results: The Traffic Light Switcher


Meet the Jammer v.1



Meet the Jammer v.1



Meet the Jammer v.1




We need a solution...

GFP Fluorescence

% o

f Max

imum

Arabinose +Arabinose -Positive Control

Negative Control

Incomplete Jammer


Meet the Jammer v.2



Meet the Jammer v.2




We found the solution

GFP Fluorescence

% o

f Max

imum

Arabinose +Arabinose -Positive Control

Negative Control

Complete Jammer



Jammer: Any Input, Any Output















Promoters


The Traffic LightDesign
















































Our ContributionsConclusions



Family of Concentration

Sensitive Promoters




Sensitive Promoters

Jammer: Modular Repression Technology




Sensitive Promoters

Jammer: Modular Repression Technology

Traffic Light Biosensor using Threshold

Detection


One more thing...


Applications: The Next Big ThingsOne more thing...

}{



Organ Rejection Biomarkers

MMP9

Off

PhysiologicalEffectors

Inhibitor Production}{

}{



Organ Rejection Biomarkers

MMP9

Off

PhysiologicalEffectors

Inhibitor Production}{

}{Environmental Homeostasis

Inorganic Iron

Iron Sequestration

Off

Iron Release }{


a BIG THANK YOU to...Acknowledgements

Teaching andLearningEnhancementFund

• Dept. of Chemical & Biological Engineering• Genome BC• Integrated DNA Technologies



Dr. Eric T. LagallyFaculty Sponsor



Dr. Eric T. LagallyFaculty Sponsor

Soon-to-be-Dr. Paul JaschkeGraduate Advisor



Alex NgAmelia Hardjasa

Calvin ChanCharles Howes

Eric MaHank Yu

Heather KempthorneJanny Ke

Karen CheshireMark Ling



Questions


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


Lock and Key Activation SystemQ&A

Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.

+


Lock and Key Activation SystemQ&A

Berkeley iGEM Team 2006Issacs et al. (2004) Nature Biotech.

+


RNAi-like pathways in ProkaryotesQ&A

RNase III degrades dsRNA

Meng et al. (2008) Methods Enzymol. 447(119-129)


Documents

E. coli Traffic Light Biosensor2009.igem.org/files/presentation/British_Columbia.pdf · 2009. 11. 16. · E. coli Traffic Light Biosensor British Columbia 2009 iGEM Team Questions