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My N.U. P.A.L.Northwestern University’s
Pseudomonas aeruginosa Locator
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1.7 million
170,000
Hospital acquired Infections
Pseudomonas aeruginosa Infections
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R
R
Target Gene
Autoinducergene
Expression
R
Quorum Sensing
Bacterium
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Quorum Sensing in Pseudomonas aeruginosa
RhlRLasR
LasPLas sequence
RhlP
LasR
LasR LasR RhlR RhlR
RhlR RhlR
PAI-1
RhlR
PAI-2
LasR
Rhl sequence
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Project GoalBiosensor
Fast Simple & Inexpensive
Reliable
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System Overview
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R
R
RBSRBS
Induced Promoter
Constitutive Promoter
R-protein synthase
GFP synthase
Autoinducer
R
R
R
E. Coli Biosensor
Pseudomonas aeruginosa
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Finding the Parts
lasR
rhlRlasP
rhlP
GFP
RBS
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Parts Submitted 38 parts
19 parts21 parts
Designed and Constructed
Function Validated
Characterized
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Parts Characterized 6 parts
4 parts5 parts
Rhl promoter Constructs
Las Promoter Constructs
Newly Isolated Promoter Constucts
4 parts Previously Existing Registry Parts
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Favorite PartsLasR, PAI-1
RhlR, PAI-2 Newly isolated
RhlR, PAI-2
BBa_K575024
BBa_K575033
BBa_K575046
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Testing Methods
Overnights Sample Preparation
Plate Reader
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RBS GFP RBS LasRConstitutive Promoter
LasR/PAI-1 Induced Promoter
BBa_K575024
LL
Las Based Detection System
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No Autoinducer
Autoinducer added
Time Response of LasR/PAI-1 BiosensorNo discrimination between different concentrations
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LasR/PAI-1 Biosensor Exhibits Binary Sensing
Autoinducer Concentrations (μM)
Steady State Fluorescence
per OD
Steady State Fluorescence of the construct does not change in
response to varying PAI-1 dosages
Flat transfer function
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RBS GFP RBS RhlRConstitutive Promoter
RhlR/PAI-2 Induced Promoter
BBa_K575033
RR
Rhl Based Detection System
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No Autoinducer
Autoinducer added
Increasing autoinducer concentration
Time Response of RhlR/PAI-2 Biosensor
Discriminates between different concentrations
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RhlR/PAI-2 Biosensor Exhibits Dose-Dependent Response
Steady State Fluorescence
per OD
Autoinducer Concentrations (μM)
Y=68.234ln(x) + 142.89R2=0.9322
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RBS GFP RBS RhlRConstitutive Promoter
RhlR/PAI-2 Induced Promoter
BBa_K575046 (Isolated from Genome)
RR
Rhl Based Detection System
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Time Response of RhlR/PAI-2 Biosensor Isolated from the Genome
No Autoinducer
Autoinducer added
Increasing autoinducer concentrationDiscriminates between
different concentrations
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Steady State Fluorescence
per OD
RhlR/PAI-2 Biosensor Isolated from the Genome Exhibits Dose-Dependent Response
Y=33.43ln(x) + 72.293R2=0.9694
Autoinducer Concentrations (μM)
Exhibited superior discrimination between varying PAI-2 dosages
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System Model
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PurposeBetter understand why the biosensors behave differently in the context of the applicationsOptimize parameters and achieve the set goalDevelop and apply a model to other pathogen sensing constructs or devices
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mRNA
Ae
CP
IP
mRNA
Ai R
D
5
97
8
M4
103
12
11
12
613
GFP
Directly translated each rate to ODEsSystem of non-linear equations
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Analysis Sensitivity
SimulationIdentify Critical Parameters
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Qualitative Mathematical Fit to the LasR Biosensor Data
50 100 150 200 250 3000
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Arbitrary Concentration
of GFP
Simulated Time (min)
0.1μM
No Autoinducer
0.5μM
1μM-100μM
Data
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Qualitative Mathematical Fit to the RhlR Biosensor Data
50 100 150 200 250 3000
0.5
1
1.5
2
2.5
3
3.5
4
Arbitrary Concentration
for GFP
Simulated Time (min)
No Autoinducer0.1μM
100μM50μM
20μM
10μM
5μM
0.5μM
Data
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mRNA
Ae
CP
IP
mRNA
Ai R
D
5
97
8
M4
103
12
1112
613
GFP
Critical ParametersLasR/PAI-1
RhlR/PAI-2
↓RhlR/PAI-2
↑LasR/PAI-1
RhlR/PAI-2 , IP +ve co-operativity
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ImplicationsIdentified system parameters which fluctuated the biosensors’ response
Can potentially optimize constructs to “tune” the sensitivityGeneralized model which mathematically represents autoinducer detection
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Application
Blue LED
PhotodiodeWell
Orange filter
Proposed Biosensor Schematic
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Application
Swab Swab is inserted into a vial with the biosensor
Biosensor fluoresces in the presence of the pathogen
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TEAR GAS
Artificial Life
Unknown consequences
BIOWARFARE
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Human PracticesWe promise not only to do the cool research, but to stand by every mistake and every error from now until the future, just like you would stand by profit taking.
-Dr. Laurie Zoloth
Dr. Dale MortensenNobel laureate in Economics
Dr. Laurie Zoloth Bioethicist
Dr. Keith TyoSynthetic biologist
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Results Summary
The Las based biosensor exhibited a binary response
The Rhl based biosensor exhibited a dose dependent responseHigh discrimination between varying autoinducer dosages exhibited by the newly isolated promoter
19 parts characterized
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Initial Goals
Simple and Inexpensive
Reliable
Fast
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Future ProspectsFuture iGEM ProjectsClinical Application
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Acknowledgements
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THANK YOU.
Questions?