Assay for phage recombination

I think I now have everything I need to assay the ability of phage to recombine in different H. influenzae competence mutants (the mitomycin C arrived yesterday), so I've gone back and looked at the procedure I used.  (Yes, I should have done this sooner...)  The procedure is a bit more complicated than I had remembered, but maybe I can streamline it.


The procedure (from my old lab notebook):

Need: 

1. Lysates of two phage strains carrying temperature-sensitive mutations in different genes (at least 10^10 phage/ml).
2. Culture of the bacteria to be tested (at least 10^9 cells/ml), in culture or competence medium.
3. Culture of wildtype cells not lysogenic for the phage, to use for the lawns.
4. Incubators at 34°C and 40°C.
5. Melted 'top agar' (0.7 % agar medium) at ~48°C.
6. Normal agar plates at room temperature.

Recombination procedure:

1. Mix:  0.3 ml cells with about 10^9 phage of each type, both phage together and each separately.
2. Incubate for 10' at 34°C.  This gives the phage time to attach to the cells and inject their DNAs.
3. Pellet the cells, resuspend them in medium, and pellet them again.  This washes away all the phage that didn't infect the cells.
4. Resuspend the cells, diluting them 1/300 in culture medium.  
5. Set aside some of this culture (room temperature) to assay for 'infectious centres'.
6. Shake this culture for 90' at 34°C.  This time allows the phage to complete one cycle of infection.  The infected cells will burst, releasing the progeny phage.
7. Add  a few drops of chloroform to the culture to kill the remaining cells, and centrifuge the culture to remove dead cells and debris.  Transfer the supernatant ('lysate') to a fresh tube.

Assaying the results:

1. Put about 0.1 ml of the wildtype culture (about10^8 cfu) into each of a series of numbered glass culture tubes, ideally at 30-34°C.
2. Dilute the phage lysates or the set-aside culture in culture medium.
3. Add 0.1 ml diluted or full-strength lysate to each culture tube.
4. Incubate 15' at 34°C.  This allows phage to attach to cells and inject their DNA.
5. Add 5 ml top agar (2.5 ml?) and pour onto the appropriate numbered plate, rocking to quickly spread the top agar before it sets.
6. Incubate the plate at 34°C or 40°C overnight.
7. Count plaques.  
8. For the lysates, the recombination frequency is the titer of plaque-forming units at 40°C divided by the titer at 34°C.  For the set-aside culture this ratio gives the proportion of infected cells that produced recombinant page.

To get the true recombination frequency requires titering the lysates, but in the old experiment I'm looking at (#125) the frequencies of infectious centres that contained recombinants were reliable indicators of the recombination frequencies in the lysates.  For log phase cells, 2 x 10^-6 of the phage in the lysate were recombinants and 5 x 10^-4 of the infectious centres contained recombinants; for competent cells the numbers were1.9 x 10^-3 and 1.7 x 10^-2 respectively.  So maybe I can just plate the infectious centres.  I'll try both the first time and see if the correlation is reproducible.

One concern with plating the infectious centres is that recombination may occur in the wildtype cells in the lawns.  The opportunity is there because every mixed-infection cell will release a mixture of phages that may multiply infect neighbouring cells.  However these cells aren't competent so phage recombination is probably not a concern.  If the background is high I could use rec- cells for the lawns.