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Biofilm Detection Using Cell-Free Approach

This project aims to characterize cell-free gene expression to detect quorum sensing in bacterial biofilms on catheters. The cell-free system eliminates reliance on bacterial chassis, making it ideal for medical devices. The sensor has specific requirements for sensitivity, signal output, lifespan, application method, temperature, and pH range. The design incorporates in vitro transcription-translation with phospholipid vesicles, cell extracts, RNA polymerase, and reporter proteins. Challenges include noise, catheter cleaning, and potential cell damage by hemolysin. Overall, this non-microbial, fast, and cost-effective detection system paves the way for future advancements.

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Biofilm Detection Using Cell-Free Approach

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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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