my n.u. p.a.l. northwestern university’s pseudomonas aeruginosa locator

My N.U. P.A.L.Northwestern University’s

Pseudomonas aeruginosa Locator

1.7 million

170,000

Hospital acquired Infections

Pseudomonas aeruginosa Infections

R

R

Target Gene

Autoinducergene

Expression

R

Quorum Sensing

Bacterium

Quorum Sensing in Pseudomonas aeruginosa

RhlRLasR

LasPLas sequence

RhlP

LasR

LasR LasR RhlR RhlR

RhlR RhlR

PAI-1

RhlR

PAI-2

LasR

Rhl sequence

Project GoalBiosensor

Fast Simple & Inexpensive

Reliable

System Overview

R

R

RBSRBS

Induced Promoter

Constitutive Promoter

R-protein synthase

GFP synthase

Autoinducer

R

R

R

E. Coli Biosensor

Pseudomonas aeruginosa

Finding the Parts

lasR

rhlRlasP

rhlP

GFP

RBS

Parts Submitted 38 parts

19 parts21 parts

Designed and Constructed

Function Validated

Characterized

Parts Characterized 6 parts

4 parts5 parts

Rhl promoter Constructs

Las Promoter Constructs

Newly Isolated Promoter Constucts

4 parts Previously Existing Registry Parts

Favorite PartsLasR, PAI-1

RhlR, PAI-2 Newly isolated

RhlR, PAI-2

BBa_K575024

BBa_K575033

BBa_K575046

Testing Methods

Overnights Sample Preparation

Plate Reader

RBS GFP RBS LasRConstitutive Promoter

LasR/PAI-1 Induced Promoter

BBa_K575024

LL

Las Based Detection System

No Autoinducer

Autoinducer added

Time Response of LasR/PAI-1 BiosensorNo discrimination between different concentrations

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

RBS GFP RBS RhlRConstitutive Promoter

RhlR/PAI-2 Induced Promoter

BBa_K575033

RR

Rhl Based Detection System

No Autoinducer

Autoinducer added

Increasing autoinducer concentration

Time Response of RhlR/PAI-2 Biosensor

Discriminates between different concentrations

RhlR/PAI-2 Biosensor Exhibits Dose-Dependent Response

Steady State Fluorescence

per OD

Autoinducer Concentrations (μM)

Y=68.234ln(x) + 142.89R2=0.9322

RBS GFP RBS RhlRConstitutive Promoter

RhlR/PAI-2 Induced Promoter

BBa_K575046 (Isolated from Genome)

RR

Rhl Based Detection System

Time Response of RhlR/PAI-2 Biosensor Isolated from the Genome

No Autoinducer

Autoinducer added

Increasing autoinducer concentrationDiscriminates between

different concentrations

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

System Model

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

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

Analysis Sensitivity

SimulationIdentify Critical Parameters

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

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

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

ImplicationsIdentified system parameters which fluctuated the biosensors’ response

Can potentially optimize constructs to “tune” the sensitivityGeneralized model which mathematically represents autoinducer detection

Application

Blue LED

PhotodiodeWell

Orange filter

Proposed Biosensor Schematic

Application

Swab Swab is inserted into a vial with the biosensor

Biosensor fluoresces in the presence of the pathogen

TEAR GAS

Artificial Life

Unknown consequences

BIOWARFARE

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

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

Initial Goals

Simple and Inexpensive

Reliable

Fast

Future ProspectsFuture iGEM ProjectsClinical Application

Acknowledgements

THANK YOU.

Questions?