Cong T. Trinh:P1transduction
P1 transduction is a technique to transfer DNA from the donor strain to the recipient strain using the virus P1. This virus facilitates the generalized transduction where the generalized transducing particles contain only the DNA of the donor strain due to the packaging error, not the DNA mixture of the donor strain and the virus. The generalized transducing particles will then infect the recipient strain and transfer their DNA through homologous recombination. There are two main steps in using P1 transduction for DNA transfer. The first step is preparing the generalized transducing particles, P1 lysates. The second step is to infect the recipient cells with the lysates.
Preparation of the P1 lysates
- i.Grow 1-3 ml of the donor strain the Z broth, overnight or from 6-8 hrs.
- ii.In a 1.5 ml tube, add 100 μL of P1 into 300 μL of the donor strain culture and incubate them at 37°C for 20 min.
- iii.Transfer the culture into the 15 mL Aerobic CULTURE tube and resuspend it in the Z broth to the final volume of 10 mL.
- iv.Incubate the culture in the shaking incubator at 37°C with a speed of 200 rpm for 6-8 hours until the clear lysates are observed. You may incubate overnight as well for lysing. To easily see the difference, a negative control of the donor strain without P1 infection can be grown in parallel.
- v.Add 100 μL of chloroform into the culture and vortex for 30 seconds.
- vi.Centrifuge the cultures at room temperature with a speed of 5,000 rpm for 10 min and carefully transfer the lysates to the 15 mL CORNING tubes.
- vii.Repeat at least once with steps v. and vi. to ensure that no cells are in the lysates.
- viii.Finally, the lysates are transferred to a new tube with an addition of 100 μL of chloroform and stored at 4°C for later use.
- i.Grow 3 mL of the recipient strain culture overnight in the Z broth
- ii.Centrifuge the culture at room temperature with a speed of 5,000 rpm for 10 min.
- iii.Resuspend cells in the Z broth and add 100 μL of P1 lysates prepared in the previous step to
bring the total volume up to 2 mL.
- iv.Incubate the culture at 37°C for 20 min.
- v.to the culture add 8 ml of citrate buffer to slow down the infection and centrifuge the culture at 4°C for 10 min.
- vi.Wash the culture twice with 10 mL of citrate buffer each through centrifugation and re-suspend as in step v.
- vii.Resuspend cells in 10 mL of LB media supplied with 25 mM of sodium citrate and incubate the culture at 37oC for 30 min.
- viii.Centrifuge the culture at 4°C with a speed of 5,000 rpm for 10 min and discard supernatant
- ix.Wash the cells twice with 10 mL of citrate buffer each through centrifugation and re-suspend as in step vii
- x.Resuspend cells to a final volume of approximately 150μL in citrate buffer and use all 150μL for spreading onto LB agar plates supplied with 2.5 mM citrate buffer and antibiotics treatment used for screening (usually Kanamycin). Incubate at 37°C overnight; growth may take 48 hours.
- xi. Once cells have grown to suitable size, make a colony streak plate using LB agar plates with appropriate antibiotics. This can be done by plucking 8 colonies from plates and streaking out. Use control of initial recipient strain on the same plate. For the construction of the mutant strain with a deletion of multiple genes, the donor strain only has a single gene deletion replaced by a kynamacin cassette, antibiotics resistance gene. If the transduction was successful, the colonies of transductants would appear on the plate after step x.
- xii. If colonies are not false positives (quite often seen as false positives) then perform colony PCR to verify if the strain was replaced with the Kanamycin cassette. Use control of wild type amplification.
- xiii. When colony PCR is successful, grow cells up to be transformed with PCP20 (antibiotic swap/temperature sensitive). Perform Heat Shock to incorporate PCP20 inside the cell and switch antibiotic cassette.
- xiv. Cure cells by removing the temperature sensitive plasmid. (growth at 37 instead of 30 and may take many rounds)
- xv. Once cured, grow cells up, extract genomic DNA and PCR to verify that ALL knockouts (yes, including the previous ones) are successfully knocked out.