My N.U. P.A.L.
Northwestern University’s
Pseudomonas aeruginosa Locator
acquired
1.7 million Hospital
Infections
170,000 Pseudomonas
aeruginosa Infections
Quorum Sensing
R
R
R
Target Gene
Autoinducer
gene
Expression
Bacterium
Quorum Sensing in Pseudomonas aeruginosa
PAI-1
PAI-2
LasR
LasR
RhlR
LasR
LasR
LasR
RhlR
RhlR
RhlR
RhlP
LasP
Las sequence
Rhl sequence
RhlR
RhlR
Project Goal
Biosensor
Fast
Simple & Reliable
Inexpensive
System Overview
Autoinducer
Pseudomonas aeruginosa
R
R
R
Induced Promoter
R
R-protein
synthase
R
RBS
Constitutive
Promoter
RBS
GFP synthase
E. Coli Biosensor
GFP
lasR
Finding the Parts
rhlP
RBS
lasP
rhlR
Parts Submitted
38 parts Designed and Constructed
21 parts Function Validated
19 parts Characterized
Parts Characterized
6 parts Rhl promoter Constructs
5 parts Las Promoter Constructs
Newly Isolated
4 parts Promoter Constucts
Existing
4 parts Previously
Registry Parts
Favorite Parts
LasR, PAI-1
BBa_K575024
RhlR, PAI-2 BBa_K575033
RhlR, PAI-2 BBa_K575046
Newly isolated
Testing Methods
Overnights
Sample
Preparation
Plate
Reader
Las Based Detection
BBa_K575024
System
LasR/PAI-1
RBS
Induced Promoter
GFP
Constitutive
Promoter
RBS
LasR
L
L
Time Response of LasR/PAI-1 Biosensor
No discrimination between
different concentrations
Autoinducer
added
No
Autoinducer
LasR/PAI-1 Biosensor Exhibits Binary Sensing
Flat transfer function
Steady State
Fluorescence
per OD
Steady State Fluorescence of the
construct does not change in
response to varying PAI-1 dosages
Autoinducer Concentrations (μM)
Rhl Based Detection
BBa_K575033
System
RhlR/PAI-2
RBS
Induced Promoter
GFP
Constitutive
Promoter
RBS
RhlR
R
R
Time Response of RhlR/PAI-2 Biosensor
Discriminates between
different concentrations
Increasing autoinducer
concentration
Autoinducer
added
No
Autoinducer
RhlR/PAI-2 Biosensor Exhibits Dose-Dependent
Response
Y=68.234ln(x) + 142.89
R2=0.9322
Steady State
Fluorescence
per OD
Autoinducer Concentrations (μM)
Rhl Based Detection
BBa_K575046
System
(Isolated from Genome)
RhlR/PAI-2
RBS
Induced Promoter
GFP
Constitutive
Promoter
RBS
RhlR
R
R
Time Response of RhlR/PAI-2 Biosensor
Isolated from the Genome
Discriminates between
different concentrations
Increasing autoinducer
concentration
Autoinducer
added
No
Autoinducer
RhlR/PAI-2 Biosensor Isolated from the
Genome Exhibits Dose-Dependent Response
Y=33.43ln(x) + 72.293
R2=0.9694
Steady State
Fluorescence
per OD
Exhibited superior discrimination
between varying PAI-2 dosages
Autoinducer Concentrations (μM)
System Model
Purpose
Better understand why the biosensors behave
differently in the context of the applications
Optimize parameters and achieve the set goal
Develop and apply a model to other pathogen
sensing constructs or devices
12
Ae
11
10
5
M
Ai
Directly
translated
each rate to
ODEs
System of
non-linear
equations
mRNA
4
R
2
1
IP
D
8
7
GFP
3
13
6
mRNA
9
CP
Analysis
Sensitivity
Identify Critical Parameters
Simulation
Qualitative Mathematical Fit to
the LasR Biosensor Data
0.7
1μM-100μM
0.6
0.5μM
Arbitrary
Concentration
of GFP
0.5
0.1μM
0.4
0.3
0.2
Data
0.1
0
50
100
150
200
Simulated Time (min)
250
300
No Autoinducer
Qualitative Mathematical Fit to
the RhlR Biosensor Data
100μM
50μM
20μM
4
3.5
3
Arbitrary
Concentration 2.5
for GFP 2
10μM
1.5
5μM
1
0.5
0
50
100
150
200
250
Simulated Time (min)
300
0.5μM
0.1μM
No Autoinducer
Data
Critical Parameters
LasR/PAI-1
Ae
10
11
12
↓RhlR/PAI-2
5
M
Ai
1 ↑LasR/PAI-1
IP
D
8
7
GFP
CP
4
R
2
mRNA
3
RhlR/PAI-2 , IP +ve
6 co-operativity
mRNA 9
13
RhlR/PAI-2
Implications
Identified system parameters which fluctuated
the biosensors’ response
Can potentially optimize constructs to “tune”
the sensitivity
Generalized model which mathematically
represents autoinducer detection
Application
Proposed Biosensor Schematic
Well
Blue LED
Photodiode
Orange
filter
Application
Swab
Swab is
inserted into a
vial with the
biosensor
Biosensor
fluoresces in
the presence
of the
pathogen
Dr. Dale
Mortensen
Nobel laureate
in Economics
Dr. Laurie
Zoloth
Bioethicist
Dr. Keith Tyo
Synthetic
biologist
Human Practices
We 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
Results Summary
19 parts characterized
The Las based biosensor exhibited a
binary response
The Rhl based biosensor exhibited a
dose dependent response
High discrimination between varying
autoinducer dosages exhibited by the
newly isolated promoter
Initial Goals
Fast
Simple and Inexpensive
Reliable
Future Prospects
Future iGEM Projects
Clinical Application
Acknowledgements
THANK YOU.
Questions?