igem: measurement techniques for pathway output noah helman lim lab may 2007
TRANSCRIPT
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iGEM: Measurement Techniques for Pathway Output
Noah Helman
Lim Lab
May 2007
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Outline Pathway outputs Measurement techniques
FACS Microscopy Western Quantitative mating assay
Comparison
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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)
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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 successpromoter GENE
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FACS
Fluorescence activated cell-sorter…
Measures fluorescence of thousands of cells in seconds.
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FACS data Measures scatter and fluorescence at
multiple wavelengths.
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FACS features High-throughput sampler Single cell data Sorting? Not ours, but possible…
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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.
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Microscopy
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are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
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Microscopy
Microfluidics
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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.
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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.
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Western example (Wikipedia)
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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.
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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
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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.
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Comparison of techniques Quantitative mating
Functional screen Huge dynamic range (106)