TestingTwo-hybridBaits

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Testing Two-hybrid Baits

How d oes this assignm ent 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 theplasmids 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 assignm ent

In this assignment, you need to think about how to use nutritional selection to select forthe 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 howreporter 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 areasin 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. Submityour 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


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Project 1: yeast transfo rmat ions

(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 ofplate 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 Type of plate

1. negative control: no plasmid (just water) 1. SC-Trp

2. positive control: no plasmid (just water) 2. YPD

3. bait plasmid (pLexA-bait) 3. SC-Trp

4. bait plasmid + pACT2.2-Raf 4. SC-Trp-Leu

5. bait plasmid + previous prey 5. SC-Trp-Leu

Explanations:

1. to ensure that our cultures are contamination free because they have a mutation and should not

grow without tryptophan2. to ensure that our yeast culture is a viable sample because it has all of the nutrients that itwould 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 cangrow in media lacking tryptophan and leucine

Projec t 1: X-gal f il ter assays(8 points)

After you transform your yeast strain with plasmid DNAs, youll test the resulting strainsto determine whether the reporter genes are being expressed. One of the reporter genes is lexA-

HIS3, 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 -galactosidaseactivity in the lab.


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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-Rafwere described in class; these plasmids are also described inIntegrated Genomics. pLexA-

Lin66-full encodes the entire C. elegansLin66 protein fused to LexA. This fusion protein binds

to LexA binding sites and activates transcription of the lexA-HIS3and lexA-lacZreporter genesby 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 Color

L40 W

L40 + pLexA-Ras W

L40 + pLexA-Lin66-full B

L40 + pACT2.2-Raf W

L40 + pLexA-Ras + pACT2.2 W

L40 + pLexA-Ras + pACT2.2-Raf B

L40 + pLexA-Lin66-full + pACT2.2 B

L40 + pLexA-Lin66-full + pACT2.2-Raf B

Project 1: growth tests

(4 points)

The purpose of the growth tests is to determine whether the lexA-HIS3reporter 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 tablebelow with the types of plates youll need for growth tests on each type of transformed cells.


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Test plasmid(s) Test plate for growth

tests

Control plate for

growth tests

bait plasmid SC-trp-his SC-trp

bait plasmid + pACT2.2-Raf or

bait plasmid + previous prey

SC-trp, -leu, -his 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 aboveyou predicted whether these strains would express -galactosidase from the lexA-lacZreporter

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) Negative control strain Positive control strain

bait plasmid L40+pLexA-Ras L40+ pLexA-lin66-full

bait plasmid + pACT2.2-Raf or

bait plasmid + previous prey

L40+pLexA+pACT2.2 L40+pLexA+pACT2.2-Raf

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TestingTwo-hybridBaits