The Phage Cookbook by Dominik Refardt
P1 phage is used to move markes between strains. Any marker that can be selected for can be moved. Or any piece of the E. coli chromosome that can be counterselected for can be moved by P1 phage.
The first step is to lyse your strain carrying your marker you want to move (phage lysis of a donor strain). The P1 strain used here is an obligately lytic phage and does not integrate into the chromosome of E. coli. That means that it lyses a cell it has successfully infected. This is where its transduction characteristics come into play: P1 not only destroys it host (and makes it produce phage particles and phage DNA), it also digests the hosts chromosome. During phage assembly, some phages pack pieces of E. coli chromosome by accident. The frequency of this event is very low, roughly 0.3% of so called "transducing phages" (phages that have packed a piece of E. coli DNA) will be generated. Because P1 packs around 80kb of DNA, it will pack around 2.2 minutes of the E. coli chromosome (which has 100 minutes). To make transduction efficient, it is thus required to maximize the phage titer of lysates. The higher titer a lysate has, the more likely transduction will happen.
Once the lysate is ready, the actual transduction can be made. Thereby the lysate is dumped onto a growing E. coli culture, and the cells get infected by the page. Once a transducing phage attaches to an E. coli cell, it will inject its DNA, but not lyse the cell, because the injected DNA is obviously E. coli DNA. The recipient cell will integrate this piece of DNA in its chromosome via homologous recombination. This means that it will integrate at the locus in the chromosome that matches the sequence of the foreign piece of DNA. Thereby ANY marker, might it be an antibiotic resistance cassette, a point mutation or any genetic alteration, that is coded onto the foreign piece of DNA will be integrated.
Grow up 1 mL of O/N culture of the donor. Dilute 1:100 in 3 x 5 mL LB in glass tube
Grow to OD 0.2-0.3
Add CaCl2 to a final concentration of 10 mM (50 µL 1M solution to 5 mL LB)
Add 1 µL, 10 µL, 100 µL of starter lysate to each tube. It is helpful to include a control tube with no lysate, which facilitates the identification of lysis.
Incubate until culture lyses (approx. 3-4 hours for 10 µL at 37°C, O/N for 1 µL at 30°C)
Use the lysate with the lowest multiplicity of infection (MOI) (1 µL), add 2 drops of chloroform (trichloromethane), vortex briefly (don’t vortex too fast, it can damage the phage tails)
Let stand at room temperature for 10 min
Spin 10 min at 5000 rpm or 15 min at 4000 rpm to remove dead bacteria
Add supernatant to glass tube with screw cap. Add two more drops of chloroform. Store at 4 °C.
This is the (Boos Lab Edition from Konstanz), thanks to T. Bergmiller.
Grow O/N culture of recipient strain
Inoculate 4 ml fresh LB 1:100 with O/N strain
Grow to mid- to late exponential phase (OD 0.5 to OD 1). Transduction will be most efficent with exponential growing cells!
add CaCl2 to 10 mM final concentration
split into 4x1 mL into Eppendorf tubes
take an aliquote of your lysate and spin it down for 1 min max speed to remove possible bacterial contamination (phages don’t pellet at that speed)
add 1, 10 and 100 µL of your centrifuged lysate, the forth tube serves as control, don’t add lysate to that
let the four tubes stand for 15 to 20 min on your bench at room temperature (infection)
stop the infection after that time by adding 20 to 50 mM sterile Sodium Citrate (also add SoCitrate to your control)
incubate at 37° for at least one hour (recombination and phenotypic expression)
spin down, remove most of the superatant, resuspend the pellet in residual supernatant and plate on selection plates containing your desired antibiotic PLUS 20 mM NaCitrate (also plate empty control!!)
additionally plate an aliquote of the lysate (10 µL) to check for possible contamination of the lysate
pick clones and streak 3x sequentially on SoCitrate plates to remove phage that were carried over from the transduction
This is the version of N. Freed. Thanks as well.
Make LB for phage infection (LBPI): 1 mL LB, 100 mM MgSO4, 5 mM CaCl2. To make 10 mL: 8.95 mL LB, 1 mL 1M MgSO4, 50 µL 1M CaCl2
Spin down 1 mL of recipient strain (mid to late exponential culture). Discard supernatant.
Resuspend in your recipient in 1 mL LBPI.
Set up the following five tubes in 5-mL culture tubes:
100 µL lysate only + 100 µL LBPI (negative control for lysate contamination)
100 µL lysate + 100 µL cells
10 µL lysate + 100 µL cells + 90 µL LBPI
1 µL lysate + 100 µL cells + 99 µL LBPI
100 µL cells + 100 µL LBPI (negative control to check plates are working)
Incubate (no shaking) at 37 °C for 20-30 minutes (phage infection)
Add 200 µL 1M NaCitrate + 1 mL LB to each of the five tubes above (stops phage infection,
Incubate w/ shaking for 1 hour at 37°C (recovery, antibiotic resistance expression)
Spin, resuspend in 100 µL LB (normal LB, not LBPI)
Plate on LB + NaCitrate (final conc of 20 mM) + antibiotic agar plates.
Streak 3x on LB + NaCitrate (final conc of 20 mM) + antibiotic plates in order to dilute out any remaining phage. To do this, pick a single colony, streak it out, the next day pick a single colony from that plate and streak it again. The next day do the same. The strain is now free of phage and is ready for use