A Rapid Induction Overexpression System for the Fission Yeast Schizosaccharomyces pombe

Materials and Reagents

1. Consumables

   1. Test tubes (Fisherbrand, catalog number: 14-961-34, or equivalent)

   2. Standard sterile serological pipettes (2, 5, 10, and 25 ml)

   3. Micropipette tips (1,000-100 µl, 200-20 µl, 20-2 µl, and 2.0-0.1 µl)

   4. 100 × 15 mm Petri dishes (VWR, catalog number: 25384-088 or equivalent)

   5. 15 and 50 ml centrifuge tubes (Corning, catalog numbers: 430766 and 430291, respectively, or equivalent)




2. Medium and growth conditions

   Standard pombe minimal glutamate (PMG) medium, pH 5.6, supplemented as necessary with 250 mg/L each of adenine, uracil, leucine, histidine, and lysine (Sabatinos and Forsburg, 2010). Bacto agar should be added at a final concentration of 2% (w/v) for preparation of solid agar medium. Leu-PMG medium has all supplements mentioned except leucine. Add thiamine as specified in the methods. Thiamine must be added after cooling the media to 45-50°C after autoclaving for preparation of PMG agar medium with thiamine.

   
   

   The base pTIN-system vectors, pTIN and pTIN7m, are available at: https://www.addgene.org/Stewart_Shuman.

   pTIN   ID#162885

   pTIN7m    ID#162886

   Your GOI can be cloned into and overexpressed from these vectors.

   
   

   1. Adenine (Sigma, catalog number: A2786)

   2. Uracil (catalog number: U0750)

   3. Leucine (Fisher, catalog number: BP385-100)

   4. Histidine (Sigma, catalog number: H5659)

   5. Lysine (Fisher, catalog number: BP386-100)

   6. Bacto agar (BD, catalog number: 214010)

   7. Thiamine hydrochloride (Sigma, catalog number: T1270)

   8. Bacto agar (BD, catalog number: 214010)




3. Yeast competent cells and transformation

   1. Tris base (Fisher, catalog number: BP152)

   2. EDTA (Sigma, catalog number: E5134)

   3. Lithium acetate (LiOAc) (Sigma, catalog number: L4158)

   4. PEG 3350 (Sigma, catalog number: P4338)

   5. Glycerol (Fisher, catalog number: BP229)

   6. Salmon sperm DNA (ssDNA) (Sigma, catalog number: D1626)

   7. LiOAc-mix (see Recipes)

   8. PEG-mix (see Recipes)

   9. 50% Glycerol/LiOAc-mix (see Recipes)

   10. Salmon sperm DNA (ssDNA) solution (see Recipes)

   11. 2,000× Thiamine hydrochloride (see Recipes)

Equipment

1. Incubator shaker (New Brunswick Innova 44 or equivalent)

2. Micropipettes (1,000-100 µl, 200-20 µl, 20-2 µl, and 2.0-0.1 µl)

3. Standard glass Erlenmeyer flasks (volume of culture should not exceed half the volume of the flask)

4. Incubators (Nuaire IR autoflow or equivalent)

5. Test tube roller (New Brunswick, TC-7 with 25 mm test tube holder or equivalent)

6. Microcentrifuge (Eppendorf 5425 or equivalent)

7. Microcentrifuge tubes (Crystalgen, catalog number: L-2052 or equivalent)

8. Glass beads (Fisher Scientific, catalog number: 11-312C or equivalent)

9. Cooling centrifuge (Thermo Scientific Sorvall BP8 with HAEMAFlex 6 swinging bucket rotor or equivalent)

10. Centrifuge bottles (Thermo Scientific, catalog number: 3120-0250 or equivalent)

Procedure

1. Generation of yeast competent cells for long-term storage:

   1. Grow 100 ml of your fission yeast strain auxotrophic for leucine in PMG medium supplemented with leucine, histidine, lysine, adenine, and uracil at 30°C till they reach mid to late-log phase (A₆₀₀ 0.5 to 0.9). Use shaking conditions of 220 rotations per minute.

      Note: Cells must be grown in medium lacking thiamine. Do not grow cells in YES medium (Sabatinos and Forsburg, 2010), which contains thiamine.

   2. Transfer cells to a 250 ml centrifuge bottle and spin cells at 4,500 × g for 5 min at room temperature. Remove supernatant, resuspend in 5 ml of sterile water, and transfer suspension to a 15 ml centrifuge tube. Spin cells at 4,500 × g for 3 min.

   3. Remove supernatant and resuspend in 2 ml of LiOAc-mix.

      Note: Resuspend using 5 ml serological pipette (or greater volume).

   4. Split sample into two 1.7 ml microcentrifuge tubes and spin cells at 4,500 × g for 2 min at room temperature.

   5. Resuspend cells in LiOAc-mix in a final volume (cells + LiOAc-mix) of 1 ml for each aliquot.

   6. Add 0.5 ml of 50% glycerol/LiOAc-mix for each aliquot and mix by inverting the tubes or gentle pipetting.

   7. Split sample into 300 µl aliquots and store in standard 1.7 ml microcentrifuge tubes at -80°C.

      Note: Each 300 µl aliquot is sufficient for four transformations. Smaller or larger aliquots can be made. Each aliquot can be thawed (on ice) and re-frozen a maximum of two times. Limit the time in between thaw and re-freeze to maintain competence. In the interim, dry ice may be used for freezing cells before transfer to -80°C. Transformation efficiency decreases with each freeze-thaw cycle (empirical observation).




2. Yeast transformation

   1. Thaw competent cells (on ice), ssDNA, and plasmid DNA (concentration of approximately 1-2 mg/ml).

   2. For each transformation, mix 3 µl ssDNA, 2 µl plasmid DNA, and 70 µl cells. A mix of ssDNA and cells can be made before making aliquots for each transformation.

   3. Incubate at room temperature for 10 min (shaking not required).

   4. Add 290 µl PEG-mix and mix by gently inverting tubes multiple times till uniform.

   5. Incubate samples at 30°C for 1 h (shaking not required).

   6. Heat shock at 42°C for 5 min.

   7. Cool samples at room temperature for 5 min (shaking not required).

   8. Spin for 1 min at 4,500 × g at room temperature.

   9. Remove supernatant and resuspend cells in 100 µl sterile water at room temperature.

   10. Gently spread sample(s) on selective agar plate(s) using glass beads and incubate at 30°C till the appearance of single colonies (3-4 days). Leucine dropout or Leu-PMG agar medium is used for selecting the pTIN-system plasmids.

   11. Patch single transformants on Leu-PMG agar and incubate at 30°C for 1-2 days. It is best to use freshly grown transformants, but agar plates may be stored at 4°C for up to 5 days.




3. Induction and expression from the pTIN-system in yeast

   1. Inoculate patched transformants in Leu-PMG medium and grow for 5-6 h at 30°C.

   2. Dilute cells in Leu-PMG or Leu-PMG with 15 µM thiamine liquid medium and grow for the requisite amount of time needed for the experiment. The mRNA is expressed to peak levels by 1 h, and half-maximal protein expression, as assayed by Pho1 (cell surface acid phosphatase) reporter activity, is achieved by 5.6 h (Garg, 2020). To achieve lower target gene expression, thiamine concentration can be varied between 0.01 and 0.3 µM. Concentrations ≥0.3 µM lead to maximal derepression of the target gene (Garg, 2020). [15 µM thiamine has been widely utilized to repress the nmt1⁺ promoter (Forsburg, 1993) and is thus used for maximal derepression from the pTIN-system]. Cells can be used for the assay of choice after the desired period of overexpression.

   3. For growth analysis, continue from step 1. Dilute cells to 0.1 A₆₀₀ and make serial dilutions (e.g., 5-fold dilution series) in Leu-PMG medium. Spot 3 µl of the serial dilutions on Leu-PMG Agar or Leu-PMG Agar containing 15 µM thiamine. Incubate plates at the desired temperatures. Incubation time can be adjusted until the desired colony size is achieved. Incubation duration must be standardized to allow the formation of similar colony sizes of wild-type cells when grown at different temperatures.

Data analysis

Representative data: Representative data and details of experiments used to validate the system are provided in the original published research article of this method (Garg, 2020). These can be accessed through the following link: https://rnajournal.cshlp.org/content/26/11/1743.long.

Recipes

Yeast competent cells and transformation

Sterilize stock solutions (10× TE [Tris-EDTA buffer], 1 M LiOAc, 50% PEG 3350, 62.5% Glycerol, and ssDNA) by autoclaving. The thiamine hydrochloride solution should be filter-sterilized.

1. LiOAc-mix

   (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM lithium acetate)

   1 ml 10× TE

   1 ml 1M LiOAc

   8 ml water

2. PEG-mix

   (40% PEG 3350, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM lithium acetate)

   8 ml 50% PEG 3350

   1 ml 10× TE

   1 ml 1 M LiOAc

3. 50% Glycerol/LiOAc-mix

   (50% glycerol (v/v), 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM lithium acetate)

   80 ml 62.5% Glycerol

   10 ml 10× TE

   10 ml 1 M LiOAc

4. Salmon sperm DNA (ssDNA)

   10 mg/ml in water

5. 2,000× Thiamine hydrochloride

   10 mg/ml in water (30 mM stock solution)

Acknowledgments

I am thankful for the support from Drs. Stewart Shuman (S.S.) and Beate Schwer (B.S.). This work was funded by National Institute of General Medical Sciences (grant number R01-GM52470 to S.S. and B.S.; R35-GM126945 to S.S.). This method was originally published in the RNA journal (Cold Spring Harbor Laboratory Press) (Garg, 2020).

Competing interests

Patent Application No.: PCT/US21/40051; International Filing Date: July 1, 2021; Title: lncRNA-regulated gene expression system; Applicant: Memorial Sloan Kettering Cancer Center; Inventor: Garg, Angad

References

1. Forsburg, S. L. (1993). Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res 21(12): 2955-2956.
2. Garg, A. (2020). A lncRNA-regulated gene expression system with rapid induction kinetics in the fission yeast Schizosaccharomyces pombe. RNA 26(11): 1743-1752.
3. Maundrell, K. (1990). nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem 265(19): 10857-10864.
4. Sabatinos, S. A. and Forsburg, S. L. (2010). Chapter 32 - Molecular genetics of Schizosaccharomyces pombe. In: Methods in Enzymology. 470: 759-795. Academic Press.