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 promoterGENE

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. 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 (10 6 )