Download iGEM: Measurement Techniques for Pathway Output

iGEM: Measurement
Techniques for Pathway
Output
Noah Helman
Lim Lab
May 2007
Outline
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Pathway outputs
Measurement techniques
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FACS
Microscopy
Western
Quantitative mating assay
Comparison
Pathway outputs
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MAPK pathways mediate cellular decisions
(mating vs. budding / proliferation vs.
differentiation)
Yeast mating pathway generates three
responses
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Gene transcription program
Cell cycle arrest
Shmooing
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(cytoskeletal rearrangement)
Qu ic kTi me™ a nd a
TIFF (Unc om pres se d) de co mp re ss or
are n ee de d to s ee th is pi ctu re .
Mating pathway
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What can we measure to
read out pathway
activity?
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Some genes are
dramatically upregulated
during alpha factor
stimulation.
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promoter
GENE
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pFus1-GFP integrated
into genome.
Phosphorylation of
pathway components
Mating success
FACS
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Fluorescence activated cellsorter…
Measures fluorescence of
thousands of cells in
seconds.
FACS data
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Measures scatter and fluorescence at
multiple wavelengths.
FACS features
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High-throughput sampler
Single cell data
Sorting? Not ours, but possible…
Microscopy
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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 (postprocessing) and follow
total fluorescence over
time.
Microscopy
QuickTime™ and a
decompressor
are needed to see this picture.
QuickTime™ and a
decompressor
are needed to see this picture.
Microscopy
Microfluidics
QuickTime™ and a
decompressor
are needed to see this picture.
Western blot
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Technique to measure proteins directly
in a population of cells
Antibodies specific to protein of interest
are used to achieve signal over bkgnd.
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Antibodies can be specific to protein, or
protein state (e.g. phospho-) or targeted to
a tag that is genetically added to the
protein.
Western: a basic technique
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Lyse cells
Add SDS and run gel
Transfer proteins onto nitrocellulose
membrane by “blotting”.
Probe with antibodies.
Detection by radioactivity, fluorescence,
etc.
Western example (Wikipedia)
Quantitative mating
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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.
Comparison of techniques
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FACS
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Many cells studied in short time.
Easy to process hundreds of strains.
Information at the single cell level.
Time courses
Microscopy
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Individual cell information, even over time lapse.
Cell morphology
Subcellular localization
Comparison of techniques
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Western blot
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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.
Comparison of techniques
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Quantitative mating
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Functional screen
Huge dynamic range (106)