Specific Question:

What other bacterial proteins are transported into the epithelial cells via type three secretion?

Experimental Design:

1) Mutagenesis

    Introduce a GFP-expressing transposon into an EPEC strain via a plate mating.

    Verify which colonies contain a mutation with UV light.

2) Type Three Secretion

    Streak out colonies and grow in broth.

    Grow epithelial cells in a 96-well plate.

    Place bacterial cultures in wells to induce type three secretion.

    Use UV light to determine which epithelial cells contain GFP.

3) Identification of mutants

    Chromosomal prep and digest.

    Southern blots.

    Gel purification.

    Sequence off the transposon.

    Compare to known sequences.

    Classify

Potential Problems

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