Biology 2581B Lecture : Lecture 21 Notes.docx

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maroonelk470

7 Apr 2013

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Genetics lecture 21 notes analyzing the code from genome to phenome. Defining the interactome (protein-protein) two hybrid assay: Separate the activities of the gal4 protein. One component contains gal4 binding domain and other contains gal4 activation domain. Create a fusion protein that expresses a protein that consists of the binding domain. When proteins don"t interact gal 4 binding domain bound to cis acting element & no activation of reporter gene. When proteins interact binding domain in close proximity to activation domain they can activate expression of reporter. Have one budding yeast strain expressing the dna binding domain to your first protein of interest b for bait. Have another haploid strain of opposite type expressing activation domain fusing to second protein of interest. Mix 2 cells together forms a diploid (induced mating) where diploid strain is expressing both fusion proteins. If 2 proteins don"t interact, then reporter is off.

Related Questions

You are a researcher who has just discovered a new protein in a fungus. To help determine the protein's function, you want to identify the other proteins in the fungal cell with which your protein interacts. How do you design a yeast two-hybrid experiment to address this problem?

a) Express the protein in bacterial strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using bacterial strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Encourage conjugation between strain 1 and strain 2, and look for colonies that express a reporter gene. These colonies will generally arise from cells containing fusion protein that interacts with your target protein.
	b)	Express the protein in yeast strain 1 as a fusion protein with the ribosomal-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.
	c)	Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express a reporter gene. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.
	d)	Express the protein in yeast strain 1 as a fusion protein with the DNA-binding domain of Gal4p. Using yeast strain 2, make a library representing all fungal proteins expressed as fusion proteins with the Gal4p activation domain. Mate strain 1 with the strain 2 library, and look for colonies that express the target gene, which creates either a color or another selective advantage. These colonies will generally arise from mated cells containing fusion protein that interacts with your target protein.

Biology 2581B Lecture : Lecture 21 Notes.docx

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