Dorman:P22 transduction

P22 is a generalized transducing phage used for constructing Salmonella strains. The P22 derivative used in our lab has a HT (high-transducing) mutation, which greatly increases the frequency of packaging Salmonella DNA into phage heads. It also has an int mutation, which prevents lysogenization of transductants.

Preparing P22 phage lysate

Day 0

1. Grow the donor strain overnight in L at 37°C in the shaking incubator.

Day 1

1. Add 10 μL of this culture to 10 mL of L (+ antibiotic as appropriate) in a 250 mL conical flask.
2. When this culture reaches an OD₆₀₀ of ~ 0.15, add 20 μL of P22 stock.
3. Grow for 4 more hours. You should see that not much more growth will occur.
4. Add 500 μL of cholorform to the flask, swirl the flask to mix, and leave on the bench for 10 min.
5. Spin down the phage for 20 min @ 4000 rpm.
6. Decant the supernatant into a clean tube, and add a few drops of chloroform.

You've got P22 that can be used for transductions.

Transduction of P22

Day 0

1. Grow up the recipient strain overnight in L.

Day 1

1. Set up multiple transductions:
   • 100 μL of P22 lysate + 100μL of recipient strain
   • 100 μL of 1:10 diluted P22 lysate + 100μL of recipient strain
   • 100 μL of 1:100 diluted P22 lysate + 100μL of recipient strain
   • 100 μL of recipient strain (no phage control- confirms that there are no mutants in the recipient population)
   • 100 μL of P22 lysate (no recipent control- confirms that the lysate is not contaminated with bacteria.)
2. Incubate these mixtures at 37°C (no shaking necessary) for 1 hour.
3. Plate the entire volume of the transduction mixtures onto L agar plates with appropriate antibiotics.

Day 2 You should have colonies on some of your plates. To minimize the possibility of working with a double transductant, use colonies of the plate that used the most diluted P22. While int P22 cannot form turn lysogens, pseudolysogens can occur. We can distinguish phage-cured transducatants from pseudolysognens by streaking on green agar plates. Phage free colonies are light green, while psedolysogens are dark blue-green.

1. Streak a desired number of colonies onto green agar plates. I typically pick four for routine strain construction.

Day 3 Examine your green plates. I typically pick one pale colony of each plate and streak again on green agar.

Day 4 At this point, all of your colonies should be pale. I pick one colony off each plate and use it to start a glycerol stock.

About green agar

This agar contains excess glucose, which is fermented by the Salmonella growing on the plate, producing large amounts of acid. In colonies of pseudolysogens, some cells will lyse, and the pH indicator in the plate will cause the agar to turn dark blue-green.

Recipe:

Green agar base:

• 8 g tryptone
• 1 g yeast extract
• 5 g NaCl
• 15 g agar

in 1 L water Autoclave.

(We get solidified green base from the prep room in 100 mL bottles.)

1. To make the plates, at 62.5 mg Alizarin yellow (on Dan's bench) to the 100 mL bottle of green base, and place the agar in the steamer.
2. When the 100mL bottle comes out of the steamer and has been cooled in the 55° bath, add 0.33 mL of 2% Aliline blue (on Dan's bench), 4.2 mL of 20% glucose, and appropriate antibiotics.