P1 transduction using P1 vir. Dorman lab. Protocol. Donor strains were grown in duplicate under standard conditions from an overnight culture (1:100 dilution) or from single colony to an OD600 0.4-0.6 in 3ml of L broth containing 5mM CaCl2 and 0.2% glucose. 100µl of P1vir was added to one of the cultures. Initial lysis was typically visible an hour after addition of the phage with lysis progressing rapidly. This resulted in clearing of the culture and accumulation of clumps of cell debris. Several drops of chloroform were added to the lysate, vortexed and centrifuged at 13’200 rpm for 3 mins to pellet cell debris and any remaining whole cells. The supernatant was then passed through a 0.45 µM filter into a fresh tube and if not used immediately, stored at 4 ºC. Recipient strains were grown overnight or to turbidity in 2ml cultures from a single colony on the day. Cells were pelleted at 6’000 rpm for 5 mins and re-suspended in 2ml L containing 10ml MgSO4 and 5mM CaCl2. The reactions were set up as follows in 2ml epindorfs; A B C D E F H Phage (µl) - 1 10 100 100 100 100 Cells (µl) 100 100 100 100 500 1000 -
Reaction tubes were mixed gently and incubated at 37 ºC for 30 mins after which 200 µl of NaCi was added to each tube using an epindorf multi-pippetor. NaCi chelates the Ca2+ ions that are required for phage adhesion and thus stops further infection. 1 ml of L was added to each tube and incubated for 1 hr to allow expression of the antibiotic resistance cassette. Cells were then centrifuged, all but ~100 µl of supernatant removed, resuspended and plated onto L agar containing the appropriate antibiotic selection. Trouble shooting. The main reason for P1 transductions not working if due to a low phage titre. Many protocols suggest 1 hr growth or growth to an OD600 of 0.2 before adding phage but lysis of a more turbid culture produces higher titres and considerably increases the yield of transformants. If no high titre phage stock is available when preparing the donor one can easily prepared as follows; lawn an L agar plate containing 5 mM CaCl2 with a strain of interest and allow to dry. Spot 10 µl of a neat P1vir stock onto each quadrant of the plate and incubate upright overnight. Zones of clearing should be visible where the phage has replicated and lysed the cells. Use a sterile spatula to scrape the layer of phage/cell debris into a 1.5 ml epindorf. Add ~200 µl of L containing 5 mM CaCL¬2 followed by a few drops of chloroform and vigorous vortexing. Centrifuge the mixture to pellet cell debris and remove the supernatant. This will be a high titre stock ideal for making new donor strains.
H-NS mutants, while not significantly growth retarded in liquid culture under standard conditions, take considerably longer to grow on plates than WT colonies. It is therefore important to leave plates of ∆hns recipients at 37ºC for 2 days after transduction. All presumptive transductants should be re-streaked on selective media and then screened for the mutant allele using PCR. It also good practice to confirm that any alleles present in the recipient but not in the donor are still present after the transduction.