Katharine Kripke

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• Katharine Kripke

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Phenotype microarrays

• High-throughput (96 or 384-well plate) analysis
  of respiration in the presence of various
  substances (nutrients, antibiotics, toxins, etc.)
• For testing metabolism, the bacteria are grown in
  minimal media supplemented with only one carbon
  source (or nitrogen, phosphorus, sulfur source,
  as appropriate)
• Reporter is tetrazolium dye that is converted
  during mitochondrial electron transport
  (well-established technology)
• Mutant (green) is compared with isogenic control
  (red) strain

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Definitions

• catabolism destructive metabolism involving
  the release of energy and resulting in the
  breakdown of complex materials within the
  organism
• anabolism the constructive part of metabolism
  concerned especially with macromolecular
  synthesis
• auxotrophic requiring a specific growth
  substance beyond the minimum required for normal
  metabolism and reproduction by the parental or
  wild-type strain ltauxotrophic mutants of bacteriagt

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Definitions

• P1 transduction bacteriophage is used to
  transfer DNA containing a resistance
  marker-inactivated gene into a recipient strain
  homologous recombination replaces the gene in the
  recipient strain with the resistance marker

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PMs used in this paper

• ES carbon catabolism
• EN nitrogen catabolism
• EPS phosphorus and sulfur catabolism
• EA auxotrophy
• ES1-3 sensitivity/resistance to toxic
  chemicals/antibiotics

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Fig. 1 Respiration pathways coupled to cell
physiology
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http//www.expasy.org/cgi-bin/search-biochem-index
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Fig. 2 PM analysis of xylA amber mutant strain
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Fig. 2 PM analysis of ynjBtn10 mutant strain
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Fig. 3 PM analysis of malFtn10 mutant strain
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Fig. 4 PM analysis of cyakan mutant strain
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Fig. 5 PM analysis of fruRtn5 mutant strain
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Fig. 6 PM analysis of phoP amber mutant strain
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Questions for authors

• Authors claim high level of accuracy and
  sensitivity of PM technology but dont
  quantitate error or noise
• Stability of kit (solutions)?

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Caveats/limitations

• Customizing for different organisms dependent on
  experimental knowledge of metabolism, etc.
• Growth rate of organism is important (may not
  work with a slow grower like tuberculosis?)
• Some conditions wont lend themselves to testing
  in this way (temperature change, cell cycle,
  infection of cells, etc.)

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Modelling prospects

• High throughput assays may help pinpoint gene
  function
• Enhance metabolic models, possibly providing
  kinetic data metabolic comparisons of different
  organisms
• Combine with metabolic pathway databases to
  validate and improve them
• Combine with gene expression experiments to
  develop better models of cell function (networks,
  connectivity)

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THE END.