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The in-veso biofilm sensor

The in-veso biofilm sensor. Introduction. Aim: Characterise cell-free gene expression Use cell-free approach to detect quorum sensing within a bacterial biofilm. Application. Detect bacterial biofilm on catheters. Motivation. Bacterial biofilms are impermeable to antimicrobial agents

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The in-veso biofilm sensor

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  1. The in-veso biofilm sensor

  2. Introduction • Aim: • Characterise cell-free gene expression • Use cell-free approach to detect quorum sensing within a bacterial biofilm

  3. Application Detect bacterial biofilm on catheters

  4. Motivation • 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

  5. Project Plan • 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

  6. Specification • 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

  7. Design • In vitro transcription-translation: • Phospholipid vesicles • S12/ S30 cell extract • RNA polymerase: • Bacteriophage T7/ SP6 RNA • Reporter: • Luciferase • GFP • Alpha-Hemolysin pores Noireaux et. al.; 2005

  8. Design

  9. LuxI AHL AHL pTet luxI pTet luxR pLux hrpR hrpS RS pHrpL luc Design Biofilm bacteria Cell-free system LuxR

  10. Problems • Noise – leakage of pLux • Cleaning the catheters after application • Alpha-hemolysin might damage human cells

  11. Summary • Non-microbial detection system • Fast, simple and cheap • Novel technique • Lays a platform for future developments

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