24 August 2021

This protocol uses a nanoluciferase-PEST reporter to measure translation rate in the budding yeast S. cerevisiae. The reporter design is based on that of Masser et al. Yeast 2016 May; 33(5): 191-200 (https://dx.doi.org/10.1002%2Fyea.3155).

Protocol

Reagents

Yeast Strain

  • BY4742 + pJB749 (integrated at HO locus, selected with hygromycin)

Wet reagents

  • yeast growth media (SC or YP plus sugar if required)
  • Promega Nano-Glo (PRN1130)

Consumables

  • 96-well 2mL deep well plate (Dot cat. no 229575)
  • 96-well u-bottom microtiter plate (Thermo 1424571)
  • 96-well PCR plate (Thermo AB-0600)
  • 8-channel pipette (100uL or 200uL)
  • 8-channel pipette (20uL) (Rainin P20 LTS)
  • Breathe-easy sealing film (Sigma Z380059-1PAK)
  • 384-well white plates (Corning CLS3574-50EA)

Instruments

  • Multimodal microplate reader (Tecan Spark 20M)
  • 96-well thermocycler
  • 30 °C shaking incubator

Procedure

  1. Grow cells
    • 3 days prior
      • streak strain from glycerol stock onto YPD plate, incubate at 30 °C
    • 2 days prior
      • pick one colony and grow in liquid YPD overnight with shaking at 30 °C
        culture can be stored at 4 °C
    • 1 day prior
      • assuming a doubling time of 85 minutes and a lag phase of 170 minutes, dilute yeast into 8 mL of fresh YPD such that it will grow to an OD600 < 0.2 the next day
      • grow at 30 °C with shaking
    • Day of morning
      • dilute to OD600 < 0.05 (such that experiment will start at OD600 = 0.1)
  2. Prepare to measure translation
    • Calculate amount of Nano-Glo reagent required (will need 10 μL per measurement)
    • Prepare 0.5x Nano-Glo reagent by first mixing lysis buffer with 1:50 reagent, then diluting 1:1 with H2O
      diluting 0.5x is not necessary, but saves money and seems to work fine
      - aliquot 10 μL reagent into 96-well PCR plates for later measurements
    • Calibrate the OD readings: Need to measure blank OD600 and calculate the pathlength of the measurement - Use multichannel to transfer 3x blank media and 3x yeast growth with known OD to U-bottom 96-well microplate - Read using JB_210712A600_whole.mth on sparkcontrol (not magellan) - method first shakes for 5 seconds (linear, amplitude=4) - then reads every well at 600 nm with flashes=30 and settle time = 500 ms
      the settle time is important to get accurate readings - Using beer-lambert law (A = _εlc
      ), calculate the pathlength (I got around 0.5 cm) and note the mean blank reading
  3. Treat cells
    • Treatment will vary by experiment. In my experiments, I have been measuring the translation of two reporters both before and after heatshock.
    • Transfer 0.25 mL yeast to 2 mL deep well plate
      • To account for variability in the luminescence readings, I always normalize my readings to a control strain. I also try to mix the order of my samples and do measurements in triplicate.
        • For instance, given reporter A and reporter B, and varying concentrations of drug I would arrange my columns like so | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |—:| — | — | — | — | — | — | — | – | | A | A | B | A | B | A | B | A | B | | B | B | A | B | A | B | A | B | A | | C | A | B | A | B | A | B | A | B |

            1 2 3 4 5 6 7 8
          A - - +10 +10 +20 +20 +40 +40
          B - - +10 +10 +20 +20 +40 +40
          C - - +10 +10 +20 +20 +40 +40
    • Cover 2mL deep well block in breath-easy film, tape to shaking platform and shake at 30 °C
  4. Measure OD and luminescence (eventually want to calculate lum_per_OD to normalize for varying cell counts between samples)
    • Using multi-channel, transfer 150 μL to 96-well U bottom microplate, read OD600 as above
    • transfer 10 μL of culture to 10 μL Nano-Glo reagent in PCR plates (make sure to pipette up and down before transferring to mix any cells that have settled, then mix again 3x after transferring)
    • Transfer 15 μL to 384-well white plate
      recommended to leave a well of blank between every well to prevent bleedthrough. Remaining wells can be filled in after a few days
    • Spin for 30 seconds in plate centrifuge
    • Incubate for 4 minutes before reading
    • Measure with e.g. JB_210712_Lum_A1toB12.mth (loop of 5 measurements, integration time = 250 ms, take average of measurements during analysis)
  5. Heat shock and measure luminescence again
    • transfer 20 μL of culture to 96-well PCR plate
    • Treat at 42 °C using thermocycler
    • Re-read luminescence as above; I re-use the OD reading from above for normalization (assumes that during treatment the number of cells has changed a small amount)