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Dispersing biofilms with engineered enzymatic bacteriophage

Dispersing biofilms with engineered enzymatic bacteriophage. Doug Tischer. The Problem with Biofilms. Increases resistant to antibiotics Pose a problem for cleaning medical devices, water pipe, food contamination from industrial devices etc. EPS (Extracellular Polymeric Substance)

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Dispersing biofilms with engineered enzymatic bacteriophage

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  1. Dispersing biofilms with engineeredenzymatic bacteriophage Doug Tischer

  2. The Problem with Biofilms • Increases resistant to antibiotics • Pose a problem for cleaning medical devices, water pipe, food contamination from industrial devices etc • EPS (Extracellular Polymeric Substance) • Polysaccharides • Proteins • Nucleic Acids • Lipids

  3. The Idea • Adhesin polymeric β-1,6-N-acetyl-D-glucosamine essential for bacterial adhesion to biofilm • DspB degrades polymeric β -1,6-N-acetyl-D-glucosamine • T3 gene 1.2 allows replication in F-plasmid-containing E. coli Figure taken from Lu, 2007

  4. Construction • T7wt • T7DspB • T7control • Φ10 promotor expresses only during infection • 10B is a capsid protein • S-tag easily detected in western blot Figure taken from Lu, 2007

  5. Results Crystal Violet Staining CFU Counts PFU Counts • No replication (no T3 gene 1.2) Inoculation • T3 gene 1.2 greatly enhances phage efficacy in F-plasmid containing E. coli • DspB enhances plaque destruction • Engineered phage is replicating • PFU = Plaque forming unit Figure taken from Lu, 2007

  6. Results cont. Treated Untreated • 4.5 orders of magnitude drop = 99.997% drop in cell count • Scanning Electron Microscope of cell plaques • Significant disruption of plaques Figure taken from Lu, 2007

  7. My thoughts Liked Disliked • Very practical application • Showed phages can be engineered with several genes of interest without hindering replication • Successful proof of principle • Relatively simple construction • No new behavioral tricks • Only protein expression • No use of premade parts

  8. References • Lu TK, Collins JJ (2007) PNAS 104:11197–11202 • Mark McCormick and Robert Mierendorf. “S•Tag: A Multipurpose Fusion Peptide for Recombinant Proteins.” Novagen Inc.

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