Lidstrom:EMS Mutagenesis

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Overview

• EMS is a liquid mutagen
  • Work in the fume hood and use special precautions!
• It tends to produce a certain type of mutations
• Following subsequent rounds of replication, the original G:C base pair can become an A:T pair (a transition mutation). (wikipedia)
  • Since stop codons are rich in ATs (amber (UAG), ochre (UAA), (UGA)) EMS mutagenesis is good for generating nonsense alleles. (Anderson 1995)

Tips

• Use enough cells that you can see the biomass pellet after spinning down the culture to remove the EMS solution.
• Use clear eppendorf tubes to hold the cells in so you can see the biomass pellet.
  • One could use opaque tubes to reduce EMS's reaction with light, but the protection from light probably isn't worth the convenience of

Health & Safety

• EMS is a liquid mutagen
  • Work in the fume hood and use special precautions!
• EMS can be slowly inactivated by mixing with a sodium thiosulfate + NaOH mixture.
• Wash the cells at least twice before plating!
  • Don't want EMS getting out of the fume hood.
• Discard waste in a specially labeled bottle.
  • Neutralize the pH before turning it into EH&S.
  • Recipes found online vary a bit, but they are about _____:
• Leave all plastic tips and gloves used for the experiment in the fume hood a day or so after use. Double bag them and discard them in the normal trash stream after that.
  • Some protocols instruct you to soak pipette tips, etc. in a sodium thiosulfate + NaOH mixture for a while before discarding.

Survival Curve

• You need to figure out how much EMS (concentration) to use and how long to expose them to it.
  • Too little EMS*time → no mutations, but too much → all the cells are dead.
• It is good to do an survival curve that correlates exposure time to cell survival.
  • For a given amount of cells and EMS, the number of viable cells will decrease with exposure time.
  • You can decide how mutated you want your cells to be when you do your selection or phenotype hunting. This kill curve will help you know what exposure time correlates to what level of mutation.
  • plate 2uL on top of plate and 20uL on bottom. This is easier than doing dilutions and should give you a good feel for the kill curve given an appropriately dilute suspension of cells.
    • These culture volumes work well for a SIP3-4 culture grown overnight on methanol from a scraping of a few dozen colonies.
  • You can check with lower resolutions (less plating across exposure times) in subsequent experiments.
• Ideally you would know what mutation rate a given survival rate correlates to, but you might not be able to tell.
  • This paper says 50% survival → 250 fold increase in mutations over background rate of mutation.
  • This paper used 10% survival rate of mid-exponential E. coli MG1655
• The efficacy of EMS is dependent on pH. I believe it is less active at high pH.

Controls

• No EMS exposure, but otherwise treated the same
  • do one for time = 0, and one for the longest EMS exposure time you use.

Sample Protocol

• Grow cells
• Weigh chemicals
  • Calculate the amount of sodium thiosulfate and NaOH you will want. Dissolve in H2O
• Wash cells
  • probably unnecessary, but not too much extra work.
• Prepare eppendorf tube(s) for the no EMS controls and the different EMS exposure times.
  • labels: "+EMS" or "no EMS"
  • use translucent tubes so you can see the cell pellet after centrifugation
• Aliquot off the no-EMS samples
• Add the appropriate amount of EMS to the + EMS cell stock.
• Aliquot into the prepared eppendorf tubes.
• For each sampling time:
  • spin down the cells
  • put waste in specially labeled waste bottle with neutralizing sodium thiosulfate + NaOH
  • wash twice
  • plate on desired plates
    • volume plated depends on how likely cells are to survive your selection or screen. You don't want empty plates or a lawn, so the volume will vary.
    • You can also consider selecting in liquid if it is appropriate for your goals.

Demo

• pictures of a kill curve
• another planning spreadsheet

Open Questions

• Should you spin down the cells to remove the culture supernatant they were growing in?
  • Yes, if:
    • it has food you don't want them to have access to after mutagenesis
    • you are concerned that EMS could react with compounds in the media
    • you aren't in a rush and want to lean toward a more consistent but time consuming workflow.
  • It is probably fine to skip this step most of the time though.

Misc Notes

• The fact that EMS is a liquid is nice for health reasons.
  • You don't have to weigh it, so you won't inhale powder or accidentally leave powder behind in the lab or on your clothes
• ENU is a similar chemical mutagen.
• UV mutagenesis is safer to perform and should be considered.
  • A similar kill curve can be tested.

The chemicals

• EMS (Ethyl methanesulfonate)
  • carcinogen!!
    • this is why it works for mutagenesis
  • liquid
  • stored at RT in Aaron's drawer
  • liquid means you don't have to weigh it on a balance (better for your health)
  • can be neutralized with an equal volume of 20% sodium thiosulfate, 1% NaOH. The reaction isn't instant. It takes hours, so leave it at least a day.
• sodium thiosulfate
  • unknown source: "The half-life of EMS in 10% sodium thiosulfate is 17 minutes at 37°C and 1.4 hours at 20°C."
  • relatively non-toxic to humans (MSDS)
  • helps neutralize EMS
  • JM bought 250g in 2014. In red chemical cabinets.
• sodium hydroxide
  • strong base, of course
  • aids neutralization of sodium thiosulfate