Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

1. Set-up

1. Before commencing any work involving microbes, sterilize the workspace using 70% ethanol. Always use the necessary PPE (lab coat and gloves).
2. Ensure that LB agar plates with 1x ampicillin, commercially available P1 phage lysate solution, chloroform, 1 M sodium citrate, glycerol, and a box of pre-sterilized plastic pipette tips and cell spreaders are close at hand.
3. Prepare sterile LB by autoclaving and use it to make three 1 mL aliquots of LB salt solution.
   1. mM MgCl₂ (952.11-2.3803 μg), 5 mM CaCl₂ (11.098 mg), 0.1-0.2% glucose (100-200 μg)
   2. mM MgSO₄ (12.0366 mg), 5 mM CaCl₂ (11.098 mg)
   3. mM sodium citrate (25.806 mg)
4. Once finished, sterilize all surfaces as well as gloves with 70% ethanol and wash hands.

2. Protocol

1. Donor Phage Lysate Preparation
   1. Prepare a 1 mL culture of donor W3110 strain E. coli in LB with 1x ampicillin grown overnight at 37 °C with aeration and shaking at 220 rpm.
   2. Dilute this overnight culture 1:100 in 1 mL of fresh LB supplemented with 10-25 mM MgCl₂, 5 mM CaCl₂, and 0.1-0.2% glucose.
   3. Grow this bacterial dilution at 37 °C for 2 hours with aeration and shaking at 220 rpm.
   4. Once these cells have reached early logarithmic growth phase (noticeable growth and slight turbidity), add 40 μL of commercially available P1 phage and leave at 37 °C with aeration and shaking at 220 rpm.
      1. Before phage addition, the measured optical density at 600 nm of these cells should be approximately 0.4 (6).
   5. Monitor cells for 1-3 hours until the culture has lysed.
      1. Lysis will result in increased cellular debris as well as a notable decrease in turbidity (i.e. cells will be considered lysed once one is able to see through the culture).
   6. Add several drops (50-100 μL) of chloroform to the lysate and mix by vortex.
      1. Chloroform sterilizes the phage lysate by killing any remaining donor cells, leaving only phage and increasing the titer of this lysate.
   7. Centrifuge lysate at 14,000 rpm for 2 minutes to remove debris and transfer supernatant to a fresh tube.
   8. Add a few drops of chloroform and store at 4 °C for no more than one day.
2. Transduction
   1. Prepare a 1 mL culture of recipient J53 strain E. coli grown overnight in LB at 37 °C with aeration and shaking at 220 rpm.
   2. Transfer 100 μL of donor phage lysate (2.1) into a 1.5 mL microfuge tube and incubate with cap open at 37 °C for 30 minutes.
      1. This incubation allows for any remaining chloroform in the P1 lysate solution to evaporate before being added to the recipient cells.
   3. Gently pellet recipient strain cells by centrifugation at 6,000 rpm for 5 minutes.
   4. Resuspend these cells in 300 μL of fresh LB containing 100 mM MgSO₄ and 5 mM CaCl₂. (P1 phage requires calcium to be infectious).
   5. Set up two reactions using the recipient bacteria cells and prepared donor phage lysate: 1) transduction reaction combining 100 μL recipient J53 strain E. coli and 100 μL donor phage lysate, and 2) negative control combining 100 μL recipient J53 strain E. coli and 100 μL of LB containing 100 mM MgSO₄ and 5 mM CaCl₂.
   6. Incubate at 37 °C for 30 minutes with shaking at 220 rpm.
   7. Add 1 mL LB and 200 μL 1M sodium citrate (pH=5.5) and incubate for 1 hour at 37 °C with shaking at 220 rpm.
      1. Citrate is used to reduce the infectivity of P1 by chelating with calcium, preventing the lysis of recipient bacteria.
      2. Incubation of this solution allows for the expression of the ampicillin resistance marker.
   8. Pellet cells by centrifugation at 6,000 rpm for 5 minutes.
   9. Resuspend cell pellets in 100 μL of LB with 100 mM sodium citrate (pH 5.5). Vortex and plate entire solution for both reactions on two LB agar plates.
      1. LB plate should have 1x Amp for the transduced sample and no Amp for the negative control.
      2. P1 phage contamination on this plate requires re-streaking before freezer stocks can be prepared.
      3. If phage is not removed, cultures grown from these colonies will not grow unless in the presence of a calcium chelator.
   10. Pick about 3-4 colonies from both plates and streak again on two LB agar plates spread with 100 μL of 1 M sodium citrate (pH=5.5).
       1. LB plate should have 1x Amp for the transduced sample and no Amp for the negative control.
   11. Incubate the plates at 37 °C overnight to allow colonies free of phage to grow.
   12. Pick colonies from both plates and use them to grow overnight cultures in 5 mL of LB at 37 °C with aeration and shaking at 220 rpm.
   13. Isolate DNA from these cultures by DNA miniprep using 4.5 mL of the total culture volume.
       1. Elute DNA using 35 μL of nuclease-free water.
       2. Measure the resulting concentration by Nanodrop. Pure DNA will generate an absorbance ratio (A_260/280) of approximately 1.8.
   14. Use the remaining 0.5 mL of each culture to prepare 1 mL glycerol stocks by making a 1:1 mixture of 100% glycerol and bacterial culture.
   15. Store bacterial glycerol stocks at -80 °C.

3. Data Analysis and Results

1. Confirmation of transduction by qPCR
   1. Prepare two qPCR master mixes for six qPCR reactions, three using qPCR primers for the ampicillin resistance gene, and the other three using qPCR primers for a housekeeping gene (14.5 μL per reaction): 12.5 μL qPCR buffer mix + 1 μL forward primer + 1 μL reverse primer.
      1. In this experiment, we used SYBR Green master mix.
      2. Housekeeping gene primers were designed to amplify a segment of DNA within the bacterial gene encoding for DNA gyrase B (7).
   2. For each qPCR reaction, combine 100 μg of DNA from each reaction (10.5 μL) with 14.5 μL of qPCR master mix.
   3. Using a qPCR machine and the thermocycling protocol listed in Table 1, amplification was measured for the ampicillin resistance and housekeeping genes for all six reactions.
   4. Cq values generated by qPCR were used to calculate the normalized transduction efficiency of the ampicillin resistance gene (Figure 3), confirming the successful transduction of the ampicillin resistance gene.
      1. The Cq value, or cycle quantification value, of a sample is the earliest PCR cycle number at which a signal exceeded the background threshold is detected. Low Cq values correspond to more target sequence, vice versa.
      2. Normalized transduction efficiency of a gene within a sample can be calculated using these Cq values by subtracting the value of the housekeeping gene from that of the target gene, generating a ΔCq value, which can be used to calculate normalized transduction efficiency by 2^(-ΔCq).

	Temperature	Time
Denaturation	94 °C	2 min
40 cycles:
Denaturation	94 °C	15 sec
Annealing, extension, and fluorescence read	60 °C or 5 °C below lowest primer Tm	1 min

Table 1: qPCR Thermocycling Protocol