CHAPTER TWELVE

Saccharomyces Cerevisiae Growth Media Jessica S. Dymond1 The High Throughput Biology Center and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Theory 1.1 YPD 1.2 SC 1.3 Minimal media 1.4 Sporulation media 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Recipe 1 Preparation of YPD 5.1 Overview 5.2 Duration 5.3 Tip 5.4 Tip 5.5 Tip 5.6 Tip 5.7 Tip 5.8 Tip 6. Recipe 2 Preparation of Synthetic Complete (Drop-out) Media 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip 6.6 Tip 6.7 Tip 7. Recipe 3 Preparation of Minimal Media 7.1 Overview 7.2 Duration 7.3 Tip Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00012-X

#

2013 Elsevier Inc. All rights reserved.

192 192 192 193 193 193 193 194 197 197 197 197 197 197 198 198 198 198 198 198 199 199 199 200 200 200 200 200 200 200 201 202 191

192

Jessica S. Dymond

7.4 Tip 7.5 Tip 7.6 Tip 7.7 Tip 8. Recipe 4 Preparation of Sporulation Media 8.1 Overview 8.2 Duration 8.3 Tip 8.4 Tip References

202 202 202 202 203 203 203 203 203 204

Abstract This protocol describes the preparation of different types of media to grow the yeast Saccharomyces cerevisiae, both on plates and in liquid culture.

1. THEORY The ability to grow yeast on different types of media allows evaluation of many phenotypes. While all strains are capable of growth on ‘rich’ media (YPD), media contents can be altered to detect auxotrophic phenotypes, drug sensitivities, and metabolic capabilities, as well as to induce cell fate changes. The most commonly used yeast media are listed here. As yeast may be cultured both in liquid and on solid medium, both alternatives are described below. Media may be prepared as a concentrated stock, and diluted with either sterile water for liquid medium or molten agarose for solid medium.

1.1. YPD YPD, also called YEPD (yeast extract, peptone, dextrose), contains all materials required for the growth of auxotrophic strains. Growth of yeast in YPD is preferred for preparation of competent cells for transformation, prior to freezing or sporulation, and for general nonselective growth (see more information about these methods on Chemical Transformation of Yeast and Storage of Bacteria and Yeast). The carbon source of YPD (dextrose) may be substituted; YPD may also be supplemented with a drug of interest.

1.2. SC Synthetic complete media can be customized with a ‘drop-out’ mix, leaving out amino acids and/or other supplements for which selective growth is required. Typically the eight most commonly selected compounds are left

Saccharomyces Cerevisiae Growth Media

193

out of the base media, which is then supplemented with those desired after sterilization. The carbon and nitrogen sources may be substituted in SC media.

1.3. Minimal media Minimal media is supplemented only with essential amino acids specific to a given strain. Failure of yeast to grow on this media suggests the presence of an unrecognized auxotrophy.

1.4. Sporulation media Diploids grown in sporulation media will enter meiosis, producing spores after 3–5 days.

2. EQUIPMENT Autoclave Magnetic stir plate Erlenmeyer flask (4000 ml) Bottles Petri plates Magnetic stir bars Glass beads Aluminum foil

3. MATERIALS All media: Agar (for plates) Sterile water (for liquid media) YPD: Yeast extract Peptone Tryptophan Dextrose Commonly used drugs (may not be required) G418 (Geneticin) Nourseothricin (clonNAT) Hygromycin Synthetic complete drop-out media and minimal media: Yeast nitrogen base, without ammonium sulfate or amino acids Ammonium sulfate [(NH4)2SO4]

194

Jessica S. Dymond

Dextrose Supplements (not all may be required): Adenine hemisulfate Alanine Arginine Aspartic acid Asparagine Cysteine Glutamic acid monopotassium salt Glutamine Glycine Histidine monohydrochloride monohydrate Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Uracil Valine Commonly used drugs (may not be required) Canavanine 5-Fluoroorotic acid (5-FOA) Sporulation media: Potassium acetate (KOAc) Zinc acetate (ZnOAc)

3.1. Solutions & buffers Part 1 YPD Common Drugs Added to YPD Amount/l media

Drug

Final Concentration

Stock

G418 (Geneticin)

200 mg ml

1

200 mg ml

1

1 ml

Nourseothricin (clonNAT)

100 mg ml

1

100 mg ml

1

1 ml

Hygromycin

200 mg ml

1

200 mg ml

1

1 ml

Drugs may be prepared and stored at

20  C

Saccharomyces Cerevisiae Growth Media

195

Part 2: SC Drop-out Media SC-8 Drop-out Mix Component

Amount

Alanine

6.0 g

Arginine

6.0 g

Aspartic acid

6.0 g

Asparagine

6.0 g

Glutamic acid monopotassium salt

6.0 g

Glutamine

6.0 g

Glycine

6.0 g

Isoleucine

6.0 g

Phenylalanine

6.0 g

Proline

6.0 g

Serine

6.0 g

Threonine

6.0 g

Tyrosine

6.0 g

Valine

6.0 g

Tip

To assemble the drop-out mix, measure all ingredients into a glass bottle, add several large glass beads, cap, and shake vigorously. This mix can be stored at room temperature for several months

Tip

Eight supplements left out of the SC-8 mix are required by commonly used auxotrophs; these include adenine, cysteine, histidine, leucine, lysine, methionine, tryptophan, and uracil. These supplements are prepared as separate solutions, and may be added to the media if necessary after autoclaving. If they are to be included in the drop-out mix, quantities are listed below

Supplements left out of SC-8 drop-out mix

Adenine hemisulfate

1.5 g

Cysteine

6.0 g

Histidine monohydrochloride monohydrate

6.0 g

Leucine

12.0 g Continued

196

Jessica S. Dymond

Lysine

6.0 g

Methionine

6.0 g

Tryptophan

6.0 g

Uracil

6.0 g

Supplement Stock Solutions Component

Final Concentration

Stock

Amount/l media

Adenine hemisulfate

400 mM

40 mM

10 ml

Histidine monohydrochloride monohydrate

300 mM

100 mM

3 ml

Leucine

2 mM

100 mM

20 ml

Lysine

1 mM

100 mM

10 ml

Methionine

600 mM

50 mM

12 ml

Tryptophan*

400 mM

40 mM

10 ml

Uracil

200 mM

20 mM

10 ml

Supplement stock solutions should be filter sterilized prior to use

Tip

Supplement stock solutions are used to augment the composition of SC-8 media if so desired. Addition of methionine to media negates the requirement for cysteine, as methionine is the precursor of an intermediate used in cysteine biosynthesis. Thus, methionine may be added to cover both methionine and cysteine auxotrophies

Tip

*Tryptophan stock solution should be stored at 4  C in a bottle wrapped in aluminum foil to reduce exposure to light

Common Drugs Added to SC Media Drug

Final Concentration

Canavanine*

60 mg ml

5-FOA**

5 mg ml

1

Stock

60 mg ml

1

*Canavanine selection must be performed in SC-Arg media **5-FOA selection must be performed in uracil-containing media

Volume/l media 1

1 ml

Saccharomyces Cerevisiae Growth Media

197

4. PROTOCOL 4.1. Preparation Prepare the SC-8 drop-out mix and supplement stock solutions. If making the sporulation media, prepare and sterilize the histidine monohydrochloride monohydrate, potassium acetate, and zinc acetate solutions.

4.2. Duration Preparation

Variable (about 1 day)

Protocol

2h

5. RECIPE 1 PREPARATION OF YPD 5.1. Overview Prepare and sterilize 1 l of YPD for culturing Saccharomyces cerevisiae.

5.2. Duration 2h 1.1 Combine the following in a 4-l Erlenmeyer flask: 10 g yeast extract 20 g peptone 0.33 g tryptophan 20 g dextrose If solid media is desired, add agar to a final concentration of 2% (20 g l 1). 1.2 Add a stir bar, and place on a magnetic stir plate. 1.3 Slowly add 1 l of water while stirring. Stir until all components are in solution. 1.4 Wrap the top of the flask with two layers of aluminum foil and place it in an autoclave tray. 1.5 Add water to the autoclave tray until 0.25–0.5 inch of water covers the bottom. 1.6 Autoclave on the liquid cycle. 1.7 After autoclaving, remove the flask and place it on a magnetic stir plate. Allow media to cool while stirring.

198

Jessica S. Dymond

1.8 If pouring plates, cool to 55  C; otherwise, allow the media to cool completely. 1.9 Add drugs, if required. Mix well. 1.10 Pour plates or store liquid media.

5.3. Tip Dextrose may be replaced with an alternative carbon source.

5.4. Tip If storing liquid media for later use, it should be autoclaved in the container in which it will be stored. Media should be mixed in a large flask, and then transferred to a sealable container to be autoclaved.

5.5. Tip It is essential to add water while stirring to prevent caking of the dextrose. If severe clumping is observed, the dextrose may be prepared first in a smaller volume with both heat and stirring, then added to the other media components.

5.6. Tip If agar is included, it will not go into solution until after autoclaving.

5.7. Tip If the media appears to be burned (i.e., there are dark solid flakes in it after autoclaving), discard it. Try making it up as follows: 1. Mix the yeast extract, peptone, tryptophan, and 450 ml water in a 1-l flask 2. Dissolve the dextrose in 50 ml water in a 250-ml flask 3. Suspend the agar in 500 ml water in a 2-l flask Autoclave the media components separately and then combine them and allow it to cool while stirring.

5.8. Tip Media may be stored at room temperature for several months. If a drug has been added, media must be stored at 4  C. Plates should be stored at 4  C to prevent them from drying out. See Fig. 12.1 for the recipe for YPD media.

Saccharomyces Cerevisiae Growth Media

199

Figure 12.1 Recipe for YPD media.

6. RECIPE 2 PREPARATION OF SYNTHETIC COMPLETE (DROP-OUT) MEDIA 6.1. Overview Prepare and sterilize 1 l of SC media lacking the eight most commonly selected supplements for culturing Saccharomyces cerevisiae.

6.2. Duration 2h 2.1 Combine the following in a 4-l Erlenmeyer flask: 1.7 g yeast nitrogen base (without ammonium sulfate or amino acids) 5.0 g ammonium sulfate 1.32 g SC-8 drop-out mix 20 g dextrose If solid media is desired, add agar to a final concentration of 2% (20 g l 1). 2.2 Add a stir bar, and place on a magnetic stir plate. 2.3 Slowly add 1 l of water while stirring. Stir until all components are in solution. 2.4 Wrap the top of the flask with two layers of aluminum foil and place it in an autoclave tray. 2.5 Add water to the autoclave tray until 0.25–0.5 inch of water covers the bottom. 2.6 Autoclave on the liquid cycle. 2.7 After autoclaving, remove the flask and place it on a magnetic stir plate. Allow media to cool while stirring.

200

Jessica S. Dymond

2.8 if pouring plates, cool media to 55  C; otherwise, allow it to cool completely. 2.9 Add the necessary supplements and/or drugs using the prepared filtersterilized stock solutions. Mix well. 2.10 Pour plates or store liquid media.

6.3. Tip Dextrose may be replaced with an alternative carbon source. Ammonium sulfate may be replaced with an alternative nitrogen source.

6.4. Tip If storing liquid media for later use, it should be autoclaved in the container in which it will be stored. Media should be mixed in a large flask, and then transferred to a sealable container to be autoclaved.

6.5. Tip It is essential to add water while stirring to prevent caking of the dextrose. If severe clumping is observed, the dextrose may be prepared first in a smaller volume with both heat and stirring, and then added to the other media components.

6.6. Tip If agar is included, it will not go into solution until after autoclaving.

6.7. Tip Media may be stored at room temperature for several months. If a drug has been added, the media must be stored at 4  C. Plates should be stored at 4  C to prevent them from drying out. See Fig. 12.2 for the recipe for synthetic complete (SC) drop-out media.

7. RECIPE 3 PREPARATION OF MINIMAL MEDIA 7.1. Overview Prepare and sterilize 1 l of minimal media for culturing Saccharomyces cerevisiae.

Saccharomyces Cerevisiae Growth Media

201

Figure 12.2 Recipe for synthetic complete (SC) drop-out media.

7.2. Duration 2h 3.1 Combine the following in a 4-l Erlenmeyer flask: 1.7 g yeast nitrogen base (without ammonium sulfate or amino acids) 5 g ammonium sulfate 20 g dextrose If solid media is desired, add agar to a final concentration of 2% (20 g l 1). 3.2 Add a stir bar, and place on a magnetic stir plate. 3.3 Slowly add 1 l of water while stirring. Stir until all components are in solution. 3.4 Wrap the top of the flask with two layers of aluminum foil and place it in an autoclave tray. 3.5 Add water to the autoclave tray until 0.25–0.5 inch of water covers the bottom. 3.6 Autoclave on the liquid cycle. 3.7 After autoclaving, remove the flask and place it on a magnetic stir plate. Allow it to cool while stirring. 3.8 If pouring plates, cool to 55  C; otherwise, allow the media to cool completely. 3.9 Add the necessary supplements for known auxotrophies using the prepared filter-sterilized stock solutions. Mix well. 3.10 Pour plates or store liquid media.

202

Jessica S. Dymond

7.3. Tip Dextrose may be replaced with an alternative carbon source. Ammonium sulfate may be replaced with an alternative nitrogen source.

7.4. Tip If storing liquid media for later use, it should be autoclaved in the container in which it will be stored: media should be mixed in a large flask, and then transferred to a sealable container to be autoclaved.

7.5. Tip It is essential to add water while stirring to prevent caking of the dextrose. If severe clumping is observed, the dextrose may be prepared first in a smaller volume with both heat and stirring, and then added to the other media components.

7.6. Tip If agar is included, it will not go into solution until after autoclaving.

7.7. Tip Media may be stored at room temperature for several months. Plates should be stored at 4  C to prevent them from drying out. See Fig. 12.3 for the recipe for minimal media.

Figure 12.3 Recipe for minimal media.

Saccharomyces Cerevisiae Growth Media

203

8. RECIPE 4 PREPARATION OF SPORULATION MEDIA 8.1. Overview Prepare and sterilize 1 l of sporulation media to induce meiosis and generate spores in Saccharomyces cerevisiae diploids.

8.2. Duration 2h 4.1 Combine the following in a 4-l Erlenmeyer flask: 100 ml 1% (w/v) potassium acetate 10 ml 0.5% (w/v) zinc acetate 3 ml 100 mM histidine monohydrochloride monohydrate 887 ml water If solid media is desired, add agar to a final concentration of 2% (20 g l 1). 4.2 Swirl to mix, or stir briefly on a stir plate. 4.3 Wrap the top of the flask with two layers of aluminum foil and place it in an autoclave tray. 4.4 Add water to the autoclave tray until 0.25–0.5 inch of water covers the bottom. 4.5 Autoclave on the liquid cycle. 4.6 After autoclaving, remove the flask and place it on a magnetic stir plate. Allow it to cool while stirring. 4.7 If pouring plates, cool to 55  C before pouring; otherwise, allow the media to cool completely. 4.8 Pour plates or store liquid media.

8.3. Tip If storing liquid media for later use, it should be autoclaved in the container in which it will be stored. Media should be mixed in a large flask, and then transferred to a sealable container to be autoclaved.

8.4. Tip Media may be stored at room temperature for several months. Plates should be stored at 4  C to prevent them from drying out. See Fig. 12.4 for the recipe for sporulation media.

204

Figure 12.4 Recipe for sporulation media.

REFERENCES Referenced Protocols in Methods Navigator Chemical Transformation of Yeast. Storage of Bacteria and Yeast.

Jessica S. Dymond

Saccharomyces cerevisiae growth media.

This protocol describes the preparation of different types of media to grow the yeast Saccharomyces cerevisiae, both on plates and in liquid culture...
445KB Sizes 0 Downloads 0 Views