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iGEM: Measurement Techniques for Pathway Output

iGEM: Measurement Techniques for Pathway Output. Noah Helman Lim Lab May 2007. Outline. Pathway outputs Measurement techniques FACS Microscopy Western Quantitative mating assay Comparison. Pathway outputs.

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iGEM: Measurement Techniques for Pathway Output

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  1. iGEM: Measurement Techniques for Pathway Output Noah Helman Lim Lab May 2007

  2. Outline • Pathway outputs • Measurement techniques • FACS • Microscopy • Western • Quantitative mating assay • Comparison

  3. Pathway outputs • MAPK pathways mediate cellular decisions (mating vs. budding / proliferation vs. differentiation) • Yeast mating pathway generates three responses • Gene transcription program • Cell cycle arrest • Shmooing • (cytoskeletal rearrangement)

  4. Mating pathway • What can we measure to read out pathway activity? • Some genes are dramatically upregulated during alpha factor stimulation. • pFus1-GFP integrated into genome. • Phosphorylation of pathway components • Mating success promoter GENE

  5. FACS • Fluorescence activated cell-sorter… • Measures fluorescence of thousands of cells in seconds.

  6. FACS data • Measures scatter and fluorescence at multiple wavelengths.

  7. FACS features • High-throughput sampler • Single cell data • Sorting? Not ours, but possible…

  8. Microscopy • Observe single cells over time in many colors (3-4). • Temperature control • Low noise camera • Automated timelapse with autofocus • Can identify individual cells in software (post-processing) and follow total fluorescence over time.

  9. Microscopy

  10. Microscopy Microfluidics

  11. Western blot • Technique to measure proteins directly in a population of cells • Antibodies specific to protein of interest are used to achieve signal over bkgnd. • Antibodies can be specific to protein, or protein state (e.g. phospho-) or targeted to a tag that is genetically added to the protein.

  12. Western: a basic technique • Lyse cells • Add SDS and run gel • Transfer proteins onto nitrocellulose membrane by “blotting”. • Probe with antibodies. • Detection by radioactivity, fluorescence, etc.

  13. Western example (Wikipedia)

  14. Quantitative mating • Functional screen: using ability to perform the actual biological function as a measure of success. • QM = comparison of mating efficiency of different yeast strains with a standardized -strain.

  15. Comparison of techniques • FACS • Many cells studied in short time. • Easy to process hundreds of strains. • Information at the single cell level. • Time courses • Microscopy • Individual cell information, even over time lapse. • Cell morphology • Subcellular localization

  16. Comparison of techniques • Western blot • Looking at actual proteins of interest in middle of “black box” of signaling (vs. GFP reporter). • Time course possible. • Can observe phospho-states of certain proteins, if antibodies exist.

  17. Comparison of techniques • Quantitative mating • Functional screen • Huge dynamic range (106)

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