HTTM : Transposon mutagenesis

(1-A) Make a 15mL LB (Diaminopimelic acid [Dap], Ampicillin [Amp], Spectinomycin [Spec]) pre-culture ( 2mL per plate minimum) of the donor strain eAC494 and incubate with agitation at 37°C overnight.

(1-B) Prepare the 96 deep-well plates for conjugation :

Preheat the deep-well plates at 60°C in a sterile incubator for 0h 10m 0s* Prepare 50mLof LB-Agar for each plate and keep it above 70°C

Using a multichannel pipette transfer 300µLof molten LB-Agar in each well of the deep-well plates, paying attention not to create bubbles by keeping the tips on the side of the wells and not dispensing all the liquid.

Let dry in a biological hood for 3 days or until well dried but not cracked. (Optional : can be placed on a heating mat set at 30°C to shorten the drying time to 2 days).

(2-A) Prepare a 500mL LB (Dap, Amp, Spec) culture of the donor strain per plate by making a 1/250 dilution of the pre-culture and incubate overnight at 37°C.

(2-B) Fill the deep-well plates with chosen medium (1.5mL per well) and inoculate each well with the recipient strains. Incubate overnight at37°C

(3-A) Pellet the donor strain by centrifugation 6000x g and discard the liquid.

(3-B) Resuspend the pellet in 10mLLB per plate.

(3-C) Dispense 100µLof concentrated donor culture into each recipient well.

(3-D) Pellet the cells by centrifugation 4000x g and remove the supernatant with the Aspir-8 + 50 µL guide.

If not using the Aspir-8 + 50 µL guide, remove all supernatant and add 50 µL of LB to each well.

(3-E) Resuspend by agitating on a shaker 900rpm and do a quick spin to recover all the cells at the bottom of the plate.

(3-F) Take 50µLfrom the resupended cells and deposit them on the dried agar at the bottom of the prepared deep-well plate. Let dry 1h 0m 0s in a biological hood and cover with a gas permeable plate seal.

(3-G) Incubate the deep-well plates 2h 0m 0s at 37°C for conjugation.

(3-H) Add 400µLof selection media to each well and resuspend by agitating on a shaker at 900rpm and do a quick spin to recover all the cells at the bottom of the plate.

(3-I) Transfert 250µL of the resuspended cells to a new deep-well filled with 1500µL of selection media (with antibiotics to select for newly obtained mutants). Cover with a gas permeable plate seal and incubate with agitation at 37°C overnight.

(3-J)/(3-K) (Optional) Using 20µL of the conjugation mix make serial dilutions and spot on selective plates to estimate the number of mutants obtained per well.

Selection markers :

• Donor strain : Dap, Amp, Spec

• Recipient : Target-dependant

• Transposon mutants : Target-dependant + Spec

Make a passage from the previous plate to a new deep-well plate filled with selective medium.

The volume of the passage (optimized to pass 3 millions mutants in E.coli ) varies from day to day :

• 200µLof day 4 (4-A)
• 100µL on day 5 (5-A), 6 (6-A) and 7 (7-A)

(7-B) (Optional) In order to have a backup in case of an issue during DNA extraction, make a glycerol stock using 150µLof the culture after the passage, and store it at-80°C .

(8-A)/(8-B) Pellet cells by centrifugation 4000x g and remove the supernatant. Aspir-8 can be used to accelerate this step. Cells are ready for DNA extraction and can be stored at -80°Cuntil ready to process.

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