Introduction

This page is a resource that lists protocols that have been used by Imperial College London in the Synthetic Yeast project. If you would prefer, the protocols, and other related materials, are available as a downloadable word document, linked here.

Chunk plasmid preps

  1. Inoculate 2YT media supplemented with the appropriate antibiotic from the -80°C glycerol stock.
  2. Typical culture volumes in 50 ml Falcon tubes:

    Chunk O/N Volume Volume P1 Volume P2 Volume P3 Spin Columns
    n1 25ml 1.25ml 1.25ml 1.75ml 5
    n2 25ml 1.25ml 1.25ml 1.75ml 5
    n3 15ml 0.75ml 0.75ml 1.05ml 3
    n4 15ml 0.75ml 0.75ml 1.05ml 3
    n5 10ml 0.5ml 0.5ml 0.7ml 2
  3. Incubate overnight in a shaking 30°C incubator.
  4. Spin down tubes (ensuring they’re properly balanced) in the tabletop centrifuge at 4000 rpm for 15 minutes.
  5. Resuspend pellets in P1.
  6. Add P2 and mix by inversion.
  7. Add N3 and immediately mix by inversion.
  8. Transfer to microtubes (850 μl per tube).
  9. Spin down at top speed for 10 minutes.
  10. Pre-warm buffer EB to 55°C in heated block.
  11. Transfer clear lysate to spin columns (750 μl per column).
  12. Spin down at top speed for 1 minute.
  13. Discard flow through and add 750 μl PE per tube.
  14. Spin down at top speed for 1 minute.
  15. Discard flow through and spin down at top speed for 1 minute.
  16. Insert columns into fresh microtubes and apply 50 μl pre-warmed EB to membrane. Incubate tubes at 55°C in heated block for 10 minutes.
  17. Elute by spinning at top speed for 1 minute.
  18. Determine plasmid yield by nanodrop.

Chunk Digests

  1. Fill in a restriction digest data sheet and digest each of the chunks accordingly.
  2. Aim to digest at least 30-40 μg of n1 and n2, 20-30 μg of n3 and n4 and 10-20 μg of n5
  3. Incubate for 1 hour per required temperature
  4. Load completed digests onto a 0.8% agarose gel and run at 116V for at least 40 minutes.
  5. Ensure that bands are well separated and only excise the correctly sized bands.
  6. Transfer bands to 2 ml microtubes, do not add more than 400 mg to each tube.
  7. Add 3 times gel volume of buffer QG to each tube and incubate at 50°C until the agarose has completely melted, vortexing every minute or so. Pre-warm EB to 50°C.
  8. Once agarose has melted, add 1X original gel volume of isopropanol to the mix and vortex.
  9. Using 1 spin column per microtube, add 750 μl sample to spin column and wait for 5 minutes.
  10. Spin columns at top speed for 1 minute and then pour flow-through back into the top of the column. Spin as before and discard flow through. Repeat process until all of the melted agarose has been passed through the columns.
  11. Add 750 μl PE per tube.
  12. Spin down at top speed for 1 minute.
  13. Discard flow through and spin down at top speed for 1 minute.
  14. Insert columns into fresh microtubes and apply 50 μl pre-warmed EB to membrane. Incubate tubes at 55°C in heated block for 10 minutes.
  15. Elute by spinning at top speed for 1 minute. Add another 50 μl pre-warmed EB to the membrane and repeat elution process.
  16. Determine chunk yield by nanodrop.

Ethanol Precipitation and Ligation

  1. Pool the chunks together with diminishing amounts of each subsequent chunk e.g. if you have 10,000 ng of n1:
    • 10,000 ng n1
    • 5,000 ng n2
    • 2,500 ng n3
    • 1,000 ng n4
    • 400 ng n5
  2. Add dH2O to chunks to make up to 300 μl then add 30 μl of 3M NaAc pH5.2 and top up with 900 μl pre-chilled 100% ethanol (If chunk volume is greater than 300 μl, adjust other volumes proportionately).
  3. Incubate at -80°C for at least an hour.
  4. Pre-chill microfuge to 4°C.
  5. Spin down tube at 4°C, top speed for 30 minutes.
  6. Carefully remove supernatant ensuring that pellet is retained.
  7. Add 400 μl 70% ethanol and mix gently.
  8. Spin down tube at RT, top speed for 10 minutes
  9. Carefully remove supernatant and air-dry pellet until excess ethanol has evaporated. Be careful not to over-dry the pellet.
  10. Resuspend pellet in 10 μl 1X T4 ligase buffer. Add 0.5 μl T4 ligase and incubate at 16°C for at least 18 hours.
  11. Incubate at 65°C for 10 minutes to denature the ligase prior to transformation.

Yeast Transformation

**For best results, PEG and LiOAc solutions should be made up freshly for every transformation**

  1. Inoculate a 3 ml YPD kan50 culture with a colony of the strain to be transformed from a freshly streaked plate. Incubate overnight shaking at 30°C.
  2. The following morning, inoculate 2x 20 ml YPD cultures in 50 ml tubes with 200 μl each of the overnight culture.
  3. Incubate shaking at 30°C, periodically checking OD by adding 100 μl culture to 900 μl YPD in a cuvette and measuring on the spectrophotometer.
  4. Boil 50 μl of ssDNA for 8 minutes and then chill on ice.
  5. When the OD is 0.5 +/- 0.1, harvest cells by spinning in the benchtop centrifuge at RT, 2000 rpm for 10 minutes.
  6. Discard supernatant and resuspend each pellet in 10 ml 0.1M Lithium Acetate (LiOAc). Pool the cells into one tube.
  7. Spin down cells at RT, 2000 rpm for 10 minutes and resuspend cells in 400 μl 0.1M LiOAc.
  8. Aliquot 4 x 100 μl into fresh microtubes. Two of these tubes will be megachunk transformations, one will be the negative control (dH2O) and one the positive control (Pre-digested marker-swapper construct).
  9. Vortex the ssDNA and add 10 μl to each tube. Vortex lightly, add 2 μl ligation/dH2O/marker-swapper and then vortex lightly again.
  10. Incubate for 30 minutes at room temperature.
  11. Make up transformation mix:
  12. Reagent Final Concentration Volume per transformation Volume for master mix (5X)
    50% PEG-3350 30% 600 μl 3000 μl
    1M LiOAc 0.1M 90 μl 450 μl
    DMSO 10% 100 μl 500 μl
    dH2O N/A 98 μl 490 μl
  13. Vortex tubes lightly and add 888 μl transformation mix. Mix gently but well.
  14. Incubate at room temperature for 30 minutes. Pre-heat heated block to 42°C.
  15. Incubate cells at 42°C for 14 minutes, mixing gently by inversion halfway through.
  16. Spin down cells at 8000 rpm for 2 minutes in a microfuge. Discard the supernatant by aspiration with a pipette.
  17. Resuspend cells in 1 ml 5mM CaCl2 and incubate at room temperature for exactly 10 minutes.
  18. For each transformation, plate out 4x250 μl onto the appropriate selective medium. Also plate out 250 μl of the positive and negative control reactions.
  19. Incubate plates for 2 days in the 30°C incubator.

Selecting for Integrants

  1. When colonies appear, count the colonies on transformation plates and ensure that the control plates show the correct result.
  2. Replica plate each transformation plates using a single velvet onto media selecting for the lost marker and then onto media selecting for the gained marker. Store the transformation plates at 4°C and incubate the replica plates at 30°C overnight.
  3. Compare colonies on the lost marker selective plate to the original transformation plate. If a colony is not present on the lost marker selective replica plate, ensure that it has grown on the gained marker selective replica plate.
  4. If a colony fits the selection criteria (e.g. for a gain of URA, loss of LEU selection the colony grows on the original URA- and replica URA- plates but does not grow on the replica LEU- plate) then streak the colony onto fresh selective plates and also inoculate a 3 ml selective media overnight culture.

PCR Tag Analysis

  1. Perform genomic prep on overnight cultures of candidate colonies. We use the GC Prep method (Scientific Reports 6, Article number: 26863 [2016] doi:10.1038/srep26863):
    • Reagents - 5% Chelex 100 (95577, Sigma Aldrich) in deionised water, acid washed glass beads (G8772, Sigma Aldrich).
    • Equipment - 1.5 ml microtubes, vortex genius 3 shaker (IKA) fitted with a universal attachment adaptor (IKA, VG3.3), Dri-Block heater (Techne), microcentrifuge.
    1. In a 1.5 ml microtube, resuspend pelleted 100 μl overnight yeast culture in 100 μl 5% Chelex 100.
    2. Add glass beads to half total sample volume and vortex mix at high speed for 4 minutes.
    3. Incubate sample at 100 °C for 2 minutes.
    4. Spin microtube at top speed in a microcentrifuge for 1 minute. The supernatant is the GC prep and can be transferred to a fresh microtube, ensuring that no Chelex 100 is carried over.
  2. PCR reactions are made up as follows:
  3. Reagent Volume
    2X GoTaq Green 5 μl
    F Primer (5 μM) 1 μl
    R Primer (5 μM) 1 μl
    gDNA 0.2 μl
    dH2O 2.8 μl
  4. The PCR cycle (GoTaq Touchdown) is as follows:
  5. Number of Cycles Temperature Time
    1X 95°C 5 minutes
    20X 95°C 45 seconds
    70->50°C (-1°C per cycle) 45 seconds
    72°C 1 minute
    20X 95°C 45 seconds
    55°C 45 seconds
    72°C 1 minute
    1X 72°C 5 minutes
    1X 4°C
  6. GoTaq Green PCR samples can be loaded directly into a gel without adding loading buffer.
  7. When performing PCRTag analysis, always make sure that you include a BY4741 control for every reaction.
  8. It is recommended to initially screen a larger number of colonies for the WT and syn tags at a single locus per chunk integrated and then thoroughly screen any colonies that pass this screening round.

Phenotypic Screening

  1. Inoculate 2 X 3 ml YPD cultures with a BY4741 colony and a colony to be assessed. Incubate shaking at 30°C until the slowest growing culture reaches an OD of 0.5.
  2. Add 100 μl of the OD 0.5 culture to a fresh microtube and add proportional amounts of the other 2 cultures to fresh tubes along with DPBS to make the final mixtures 100 μl l of OD 0.5 culture.
  3. Spin down cells at 8000 rpm for 2 minutes and resuspend pellets in 100 μl l DPBS. Repeat centrifugation and resuspension in DPBS.
  4. Serially dilute the cells so that you have at least 60 μl each of neat, x10-1, x10-2, x10-3, x10-4 and x10-5 cell dilutions.
  5. Spot 10 μl of each dilution onto 3 x YPD (glucose) plates and 1 x YPEG (ethanol and glycerol) plate as shown below:
  6. Incubate:
    • 1xYPD plate at 30°C for 2 days
    • 1xYPD plate at 37°C for 2 days
    • 1xYPD plate at room temperature for 3 days
    • 1xYPEG plate at 30°C for 4-5 days
  7. Following incubation, photograph plates in the gel dock. Compare the sizes of well defined colonies for each strain (usually at the x10-4 dilution) using the contour and volume analysis tools of the Quantity One software.
  8. Perform a more thorough phenotypic screen with a wider variety of media and conditions every 4 integration cycles or so.

CRISPR Debugging

If integration of a megachunk introduces a fitness defect, a good way of isolating the cause of the defect is to replace sections of the synthetic DNA with WT versions. CRISPR can be targeted to synthetic PCRTags whilst supplying PCR product of the surrounding WT sequence as repair DNA. Sequence replacements that result in the fitness defect reverting to a WT growth phenotype are likely to have replaced the section of DNA causing the problem. The Ellis Lab yeast CRISPR protocol can be found at benchling.com/pub/ellis-crispr-tools.