P1 Transduction

Strains: Donor E. coli (eg. Keio knockout), Recipient E. coli (eg. K12), P1 phage stock (high titer)

Reagents: NZCYM media, 1M Sodium Citrate, 10mM CaCl₂, antibiotic stocks, P1 salts solution (LB with 10mM CaCl₂ and 5mM MgSO₄), LB plates

Time required: 2-3 days (excluding day 0)

A. Donor lysate preparation

Day 0

1. Inoculate a 3ml overnight culture of donor strain in LB supplemented with antibiotics (eg. Kan for keio collection donors).

Day 0

2. Inoculate a 3ml overnight culture of recipient strain in LB. (For part B)

Day 1

3. Dilute saturated donor culture 1:100 into 3mL NZCYM supplemented with 10Mm CaCl₂ and antibiotic if required (eg. Kan for keio collection donors). Incubate at 37C and start checking for cells after 40 minutes.
   • Make an additional such dilution to visually check for phage clearing later. No P1 phage will be added to this control culture.
   • Start a 42C water bath for incubation after addition of P1 phage.

Day 1

4. After around 45-60 minutes, if the culture has swirls when held up the light and shaken, add 50uL of the high-titer P1 phage stock to the culture. Incubate at 42C for 1 hour.

Day 1

5. After 60 minutes, check if the culture with P1 phage has started to clear (compared to control). If so, move the culture to 37C for 2 hours.

Day 1

6. Transfer lysed culture to a 15mL conical centrifuge tube and add 200uL CHCl₃. Vortex the tube vigorously to lyse any remaining cells and incubate at 37C for another 5 minutes.
   • At this point, start fast-cooling the centrifuge to 4C.

Day 1

7. After 5 minutes, centrifuge the 15 mL conical tubes at 9200g for 5 mins (or at max speed for 10 minutes).
   • Collect syringes and 0.45um filters for filtering the lysate.
   • Label new tubes that will be used to store the lysate.

Day 1

8. After centrifugation, carefully transfer the supernatant to the syringe and filter it through the 0.45um filter into its final tube. This P1:Donor lysate can now be stored at 4C.
   • Discard the chloroform contaminated tubes in biohazard bins.

B. Recipient transduction

Day 1 or 2

1. Pellet 1mL of saturated recipient culture for 2 minutes at maximum speed.

Day 1 or 2

2. Resuspend the cells in 500uL P1 salts solution. Transfer 100uL of these cells to 3 tubes (100uL each).
   • One of these tubes is a no-phage control and gets no P1:Donor lysate.
   • Add 10uL P1:Donor lysate to one of the other tubes.
   • Add 100uL P1:Donor lysate to the final tube.

Day 1 or 2

3. Incubate the tubes at 37C without shaking (desk top heater w/ water in the wells) for 30 minutes to allow the phage to adsorb to the cells.

Day 1 or 2

4. After 30 minutes, add 1mL of LB and 200uL of 1M Sodium Citrate to each tube and incubate for 1 hour with shaking.
   • Spread plain LB plates with 5mM Sodium Citrate (125uL of 1M for a 25uL plate) and any antibiotics required for selection of the transduced strain. (eg. Kan for keio collection donors).

Day 1 or 2

5. After 1 hour, spin down the cells, discard the supernatant, and resuspend in 100uL LB media. Spread, the cells on LB plates containing 5mM Sodium Citrate and antibiotic. Incubate the plates at 37C

Day 2 or 3

6. Check the plates the next day, no-phage plates should have no colonies. Restreak colonies from the plates made with P1:Donor lysate transduced cultures onto selective plates. You can directly inoculate these colonies (after restreaking) for step 2 of FLP recombination.