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 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

FACS

Fluorescence activated cell-sorter…

Measures fluorescence of thousands of cells in seconds.

FACS data Measures scatter and fluorescence at

multiple wavelengths.

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

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.

Microscopy

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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.

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.

Western example (Wikipedia)

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.

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

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.

Comparison of techniques Quantitative mating

Functional screen Huge dynamic range (106)