Transcript Artificial cells

The in-veso
biofilm sensor
Introduction
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Aim:
Characterise cell-free gene expression
 Use cell-free approach to detect quorum sensing
within a bacterial biofilm
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Application
Detect bacterial biofilm on catheters
Motivation
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Bacterial biofilms are impermeable to
antimicrobial agents
Biofilm-producing MRSA strains are associated
with nosocomial infection – this is a potential
future application
Cell-free approach eliminates need for bacterial
chassis
Attractive for detecting biofilms on medical
devices
Project Plan
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Proof of concept:
To model quorum sensing in biofilm using E.coli
LuxI/LuxR system
 To control the concentration of the extracellular
AHL signal
 To design a cell-free system that detects and reports
the AHL signal
 To compare the cell-free detection system with
E.coli detection system
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Specification
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The cell-free detection system should:
Have sensitivity to AHL ~100ng/ml
 Output a signal visible to naked eye within 3 hours
 Have a lifespan of 4 days
 Be applied through a gel spray or cream
 Work within temperatures between 20-30˚C
 Work in a pH range of 6-8
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Design
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In vitro transcription-translation:
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RNA polymerase:
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Phospholipid vesicles
S12/ S30 cell extract
Bacteriophage T7/ SP6 RNA
Reporter:
Luciferase
 GFP
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Alpha-Hemolysin pores
Noireaux et. al.; 2005
Design
Design
Biofilm bacteria
AHL
LuxI
pTet
luxI
AHL
Cell-free system
LuxR
pTet
luxR
pLux hrpR
hrpS
RS
pHrpL
luc
Problems
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Noise – leakage of pLux
Cleaning the catheters after application
Alpha-hemolysin might damage human cells
Summary
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Non-microbial detection system
Fast, simple and cheap
Novel technique
Lays a platform for future developments