UV mutagenesis of Bacteria

Determination of Optimal UV treatment

This procedure is used to determine optimal treatment which will be used for library generation.

A very important consideration before beginning this protocol is that you need to fully dose each condition in one go. If you partially dose cells with UV and give them time to recover, the cells will activate their OxyR regulon and be resistant to further UV.

  1. Grow culture overnight as appropriate for strain.
  2. Pellet 1mL of overnight culture at 3k rcf for 5 minutes.
    • see note 1
  3. Aspirate media.
  4. Resuspend in 1mL sterile saline.
  5. Label sterile petri dishes for each condition to be tested.
    • see note 2
    • Typical conditions (all in J/cm2):
      • 0 -- required for determining death rate!
      • 5,000
      • 10,000
      • 15,000
      • 20,000
      • 25,000
      • 30,000
  6. Near the UV crosslinker to minimize contamination concerns, transfer 120l of cells to the center of each petri dish. Dispense the suspension slowly and be careful so that all droplets have the same surface area and structure. Cells should form a distinct droplet. This way they will all have controlled conditions while being exposed to UV.
    • Work with only one droplet of suspension at a time.
  7. Place the dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish, otherwise it will block the UV from hitting the cells.
    • It is good practice to place the 0J/cm2 sample in the crosslinker as well.
  8. Transfer 100L from the droplet to a labeled tube. Some careful pipetting can increase number of cells recovered as cells may settle during experiment.
  9. Plate for ~200 cells assuming 0 death rate.
    • Replicate plating can be used to increase confidence in death rate.
  10. Grow plate overnight.
  11. Calculate death rate based on number of colonies.
    • see note 3
  12. Update following table for future reference.

Expected Results

Table of previously determined optimal (~95-99.9% death ratios).

J/cm2 Strain Species Description Researcher
27,500 SKO16 Eschericha coli BWA25113 with plasmid DED -- via SKO
37,500 BL21 Eschericha coli BL21 with YFP and kanR knocking out lacZ in chromosome TKD
10,000 ADP1 Acinetobacter baylyi wild-type strain BR

Please update with additional results!

Library Generation

  1. Grow culture overnight as appropriate for strain.
  2. Pellet 1mL of overnight culture at 3k rcf for 5 minutes in each of 4 eppendorf tubes.
  3. Aspirate media.
  4. Resuspend each in 1mL sterile saline.
  5. Label 2 sterile petri dishes with 0 and optimal J/cm2 treatment for each strain.
  6. Near the UV crosslinker to minimize contamination concerns, transfer a total of at least 11 120l droplets of cells distributed around the dish. Typically up to 6 droplets can easily be kept distinct on a single plate.
  7. Place each dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish.
    • It is good practice to place the 0 sample in the crosslinker without dosing.
  8. Transfer 100L from each droplet to a labeled tube. Some careful pipetting up and down can lead to recovery of more cells as they may settle during experiment. Take care to not introduce air bubbles.
  9. Pellet cells at 3k rcf for 5 minutes.
  10. Resuspend in appropriate growth media.
  11. Transfer appropriate amount of cells to overnight culture. This can either be a dilution or nearly the entire volume of cells depending on how large of a library you want to start with.
  12. Plate both treated and control for ~200 cells assuming 0 death rate to estimate total number of viable mutants.
    • replicate plating or higher number of cells can be used to increase confidence in death rate.
  13. Grow plate and culture o/n.
  14. Calculate number of mutants based on number of colonies.
    • This is expected to be very similar to previously calculated death ratios x the total number of cells treated.
  15. Freeze downs should be created for overnight culture of viable cells and possibly resuspended treated cells depending on downstream applications.

Notes

  1. Single attempt at using 1/3 of the total concentration of cells did not result in any noticeable differences. DED SKO GFP project.
  2. Not suggested to extrapolate kill ratios, best to test additional conditions if 95+% death rate not achieved. The response to irradiation is generally not linear.
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Contributors to this topic Edit topic TylerDeJong, DanielDeatherage, JeffreyBarrick
Topic revision: r5 - 2024-06-05 - 03:03:39 - Main.TylerDeJong
 
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