BIOL 2004

9/12/13

Testing Two-hybrid Baits

How does this assignment relate to the course?
In Project 1, youll introduce plasmids into yeast cells in order to answer the following questions: Does the bait alone activate the reporter genes? Does the bait interact with Raf to activate the reporter genes? Does a prey isolated by previous Biol 2004 students interact with the bait? In order to perform this experiment correctly and interpret your results, you need to understand how the two-hybrid system works, which proteins will be expressed from the plasmids you introduce, and what the consequences of expressing those proteins will be. This assignment will help you think about the tests you need to perform with your yeast cells and plan exactly how to do them.

Your assignment
In this assignment, you need to think about how to use nutritional selection to select for the plasmids you introduce into the yeast cells, and what are the appropriate plates to use for growth tests on those transformed cells. You also need to be able to predict whether the reporter genes will be expressed or not in yeast cells containing different plasmids. Understanding how reporter gene expression is regulated will allow you to choose appropriate control strains for your growth tests and X-gal filter assays. Proper controls are critical to make sure the tests are working properly and your results are valid.

What to hand in
You can work on this assignment with your group. Fill in the blank areas in the document below and save it with a name of the following format: 012_49_Testing_Baits, ie: your section number, followed by your group number, followed by the assignment title. Submit your document (in either Word or pdf format) through the Moodle site as before. The assignment is due by 11:55PM on Friday September 20. It will be graded out of 20 points.

Names of group members

Section number: Group number: Name of group member % contribution to this assignment

BIOL 2004

9/12/13

BIOL 2004

9/12/13

Project 1: yeast transformations

(4 points) To obtain the yeast strains you need for Project 1, you need to transform your strain with plasmid DNA(s). Positive and negative control transformations are included as part of the transformation procedure to verify that nutritional selection is operating correctly. The type of plate to use for plating each transformation is shown in the table. Explain why these are the appropriate plates to use. Plasmid(s) transformed into cells 1. negative control: no plasmid (just water) 2. positive control: no plasmid (just water) 3. bait plasmid (pLexA-bait) 4. bait plasmid + pACT2.2-Raf 5. bait plasmid + previous prey Explanations: 1. to ensure that our cultures are contamination free because they have a mutation and should not grow without tryptophan 2. to ensure that our yeast culture is a viable sample because it has all of the nutrients that it would need to help narrow down issues 3. to select for yeast cells that have taken up the pLexA plasmid which allows the cells to make the tryptophan they need to grow 4. and 5. to ensure that we have cells that have taken up both the bait and prey plasmids and can grow in media lacking tryptophan and leucine Type of plate 1. SC-Trp 2. YPD 3. SC-Trp 4. SC-Trp-Leu 5. SC-Trp-Leu

Project 1: X-gal filter assays

(8 points) After you transform your yeast strain with plasmid DNAs, youll test the resulting strains to determine whether the reporter genes are being expressed. One of the reporter genes is lexAHIS3, which youll evaluate in growth tests (see below). The other is lexA-lacZ, which allows the cells to produce -galactosidase. The X-gal filter assay allows you to detect -galactosidase activity in the lab.

BIOL 2004

9/12/13

The strains listed in the table below are potential positive or negative control strains to use for your X-gal assays and growth tests. pLexA, pLexA-Ras, pACT2.2 and pACT2.2-Raf were described in class; these plasmids are also described in Integrated Genomics. pLexALin66-full encodes the entire C. elegans Lin66 protein fused to LexA. This fusion protein binds to LexA binding sites and activates transcription of the lexA-HIS3 and lexA-lacZ reporter genes by itself (ie: it autoactivates transcription of the reporter genes). What color would you expect each of the following strains to be in an X-gal filter assay, blue or white? Yeast strain L40 L40 + pLexA-Ras L40 + pLexA-Lin66-full L40 + pACT2.2-Raf L40 + pLexA-Ras + pACT2.2 L40 + pLexA-Ras + pACT2.2-Raf L40 + pLexA-Lin66-full + pACT2.2 L40 + pLexA-Lin66-full + pACT2.2-Raf Color W W B W W B B B

Project 1: growth tests

(4 points) The purpose of the growth tests is to determine whether the lexA-HIS3 reporter gene is being expressed or not. From this information you can draw conclusions about whether a bait autoactivates or a bait and prey interact with each other. What types of plates will you use for each of your growth tests? The test plate should differ from the control plate by only a single nutrient (the one youre testing). The control plate should maintain selection for the plasmid(s) the strains carry, but should allow the strains being tested, as well as the positive and negative control strains (see below), to grow. Fill in the table below with the types of plates youll need for growth tests on each type of transformed cells.

BIOL 2004

9/12/13

Test plasmid(s) bait plasmid bait plasmid + pACT2.2-Raf or bait plasmid + previous prey

Test plate for growth tests SC-trp-his SC-trp, -leu, -his

Control plate for growth tests SC-trp SC-trp, -leu

Project 1: control strains

(4 points) The strains listed above under Project 1: X-gal filter assays could potentially be used as positive or negative control strains for your growth tests and X-gal assays. In the question above you predicted whether these strains would express -galactosidase from the lexA-lacZ reporter gene. Now consider how you expect these strains to behave in a growth test, and decide which strains would be appropriate positive and negative control strains to use in Project 1. The negative control strain should grow on the control plate used for the growth test but not on the test plate. The positive control strain should grow on both plates. In the table below, enter the best control strains to use with each set of transformants youll be testing. Test plasmid(s) bait plasmid bait plasmid + pACT2.2-Raf or bait plasmid + previous prey Negative control strain L40+pLexA-Ras L40+pLexA+pACT2.2 Positive control strain L40+ pLexA-lin66-full L40+pLexA+pACT2.2-Raf