Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

All experiments using recombinant DNA and BSL-2 organisms were reviewed and approved by the Quinnipiac University Institutional Biosafety Committee.

1. Preparation of B. burgdorferi culture for the production of φBB-1

1. Prepare Barbour-Stoenner-Kelly medium supplemented with 6.6% heat-inactivated normal rabbit serum (BSK)¹⁵. For 1 L of 1x BSK, combine the components listed in Table 1 in 900 mL of water, adjust the pH to 7.6 using 1 N sodium hydroxide, and mix slowly at 4 °C for 2-4 h. After mixing is complete, check and readjust the pH to 7.6 if necessary, and increase the volume to 1 L with water. Sterilize the medium by passing through a 0.22 µM filter (see Table of Materials) and use fresh or store at 4 °C for ≤2 months.
2. Three to five days prior to beginning the transduction protocol, inoculate 150 µL of the appropriate B. burgdorferi clone(s) into 15 mL of 1x BSK in tightly capped sterile conical centrifuge tubes (see Table of Materials). Supplement the medium with the appropriate concentration of antibiotics or combination of antibiotics for the selection and maintenance of heterologous DNA within the B. burgdorferi clone(s) (Table 2).
   NOTE: B. burgdorferi is a biosafety level 2 organism. Take all appropriate precautions while working with this organism. Perform all work with live cultures of B. burgdorferi in a certified and properly disinfected class II biosafety cabinet. Properly dispose of all material that contacts B. burgdorferi and the organisms themselves based on CDC guidelines³⁴.
3. Incubate the cultures at 33 °C without shaking until the cultures reach a density of ≥5 × 10⁷ spirochetes·mL⁻¹, which takes approximately 3-5 days.

2. Determine the density of the B. burgdorferi culture(s) (modified from Samuels)¹⁵

1. For densities anticipated to be above 5 × 10⁶ cells·mL⁻¹, determine the cell density using spectroscopy.
   1. Transfer 1 mL of culture to a 1.7 mL microcentrifuge tube and centrifuge at 8,000 x g for 5 min at room temperature.
   2. Discard the supernatant and resuspend the cell pellet in 1 mL of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄). Transfer the entire cell suspension to a semi-micro UV transparent cuvette.
   3. Determine the optical density of the resuspended sample at a wavelength of 600 nm (A₆₀₀). Zero the spectrophotometer (see Table of Materials) against PBS.
   4. To calculate the concentration of spirochetes·mL⁻¹ in the original culture, multiply the optical density at A₆₀₀ by 1.4 × 10⁹.
2. For densities anticipated to be between 5 × 10⁴ cells·mL⁻¹ and 5 × 10⁶ cells·mL⁻¹, determine the cell density using a Petroff-Hausser counting chamber (see Table of Materials) to directly count the number of spirochetes. This method also can be used for higher densities following appropriate dilution.
   NOTE: The visualization of live spirochetes requires a microscope modified with a darkfield condenser.
   1. Apply 10 µL of sample to the counting chamber and cover with appropriate cover glass. For densities higher than 1 × 10⁷, dilute the sample in PBS to yield 50-100 spirochetes per field.
   2. Count the cells using a darkfield microscope at a magnification of 200x-400x. Count the entire field of 25 groups of 16 small squares in all planes.
   3. Multiply the number counted by the dilution factor (if any) and 5 × 10⁴ to yield cells·mL⁻¹ of original culture.

3. Induction of B. burgdorferi phage φBB-1

NOTE: Sterilize all the glassware and plasticware by autoclaving; sterilize all the solutions by autoclaving or filtration through a 0.22 µM filter. The steps below are presented based on volumes of 15 mL, but the method is scalable to smaller or larger volumes depending on the individual needs of the experiment.

1. For the B. burgdorferi culture from which phage will be produced (the donor), use the concentration calculated by either method in step 2 to determine the volume of starter culture needed to yield 4 mL of 2 × 10⁸ spirochetes·mL⁻¹. This will yield a final concentration of 5 × 10⁷ spirochetes·mL⁻¹ in 15 mL during the recovery stage (step 3.6 below).
2. Centrifuge the volume of culture calculated in step 3.1 at 6,000 x g for 10 min. Decant the supernatant and resuspend the pellet in 4 mL of fresh BSK. Transfer the sample to the smallest sterile tube available to hold the sample with minimal head space.
3. Add the appropriate amount of inducing agent to the recommended concentration (Table 3) based on a culture volume of 4 mL to induce phage production. Cap the tube tightly and mix thoroughly.
4. Incubate the sample at 33 °C for 2-4 h.
5. After incubation, transfer the sample to a 15 mL centrifuge tube. Centrifuge the sample at 6,000 x g for 10 min. Decant the supernatant.
6. Resuspend the cell pellet in 15 mL of 1x BSK.
   NOTE: After induction of the phage, there are two different ways to proceed with the transduction assay. These methods are presented in Figure 1 and in step 4 and step 6.

4. Transduction during co-culture following exposure of the donor to the inducing agent (Figure 1A)

NOTE: This protocol can only be used when the phage-producing strain (donor) has resistance to a particular antibiotic and the strain to be transduced (recipient) has resistance to another antibiotic.

1. Prepare a B. burgdorferi culture to be used as the recipient in transduction assays, as described for the donor strain in step 1 above. Based on the density determined as in step 2, calculate the volume needed to yield 15 mL of 1 × 10⁷ spirochetes·mL⁻¹.
2. Centrifuge the volume of culture calculated in step 4.1 at 6,000 x g for 10 min. Decant the supernatant.
3. Resuspend the pellet in 1 mL of culture from step 3.6 (resuspended phage donor). Add the resuspended recipient back into the culture with the donor. Do not supplement with antibiotic. The total volume containing both cultures is 15 mL.
4. Incubate at 33 °C for 72-96 h.
5. Perform selection of transductants by solid-phase plating after co-culture as described in step 7.

5. Polyethylene glycol (PEG) precipitation to recover phage for use in transduction assay

NOTE: This protocol can be used in cases where the phage-producing strain (donor) has resistance to a particular antibiotic and the strain to be transduced (recipient) either has no antibiotic resistance or resistance to another antibiotic.

1. Supplement the culture from step 3.6 with the appropriate antibiotic at the concentration indicated in Table 2. Incubate the sample at 33 °C for 72-96 h.
2. Prepare solutions for PEG precipitation.
   1. Prepare 500 mL of 5 M NaCl. Sterilize by autoclaving and let cool prior to use. Store at room temperature.
   2. Prepare 500 mL of 40% PEG by dissolving 200 g of PEG8000 in 400 mL of water; heat gently while stirring until the solution is well-mixed. Bring the volume up to 500 mL with water. To completely dissolve the PEG8000 and sterilize the solution, autoclave the solution and cool prior to use. Store at room temperature.
   3. Prepare 100 mL of suspension medium (SM; 100 mM NaCl, 10 mM MgSO₄, and 50 mM Tris-HCl [pH 7.5]). Sterilize by autoclaving. Store at 4 °C.
3. For PEG precipitation of phage from the donor B. burgdorferi clone (from step 3.6), after 72-96 h of incubation, centrifuge the samples at 8,000 x g for 20 min at 4 °C.
4. Decant the supernatant into a clean 50 mL conical tube; dispose of the cell pellet. Add 5 M NaCl to a final concentration of 1 M. Mix well. Rock gently at room temperature for 1 h.
5. Centrifuge the samples at 8,000 x g for 10 min at 4 °C. Decant the supernatant into a clean 50 mL conical tube; the pellet might be small or absent. Add 40% PEG8000 solution to the supernatant to a final concentration of 10%. Mix well and set on ice for more than 1 h up to overnight.
   NOTE: Longer times do not seem to correlate with significantly increased phage recovery.
6. Centrifuge the samples at 8,000 x g for 20 min at 4 °C. Discard the supernatant and remove as much excess liquid as possible without losing any pellet, which contains the phage particles.
7. Resuspend the pellet in a minimal volume of SM, using the SM to wash down the side of the bottle and collect any potential phage particles. The recommended ratio is 400 µL of SM per 10 mL of original supernatant, but depending on the size of the pellet, more or less SM may be required for complete resuspension.
8. Treat the recovered phage sample with an equal volume of chloroform based on the volume of resuspension. Mix the sample well and then centrifuge at 8,000 x g for 10 min. Remove the aqueous (top) layer to a clean tube, avoiding any of the thick interface layer.
   NOTE: φBB-1 is a non-enveloped bacteriophage and is not susceptible to chloroform treatment²⁵. This step is done to further disrupt any membrane-bound structures (i.e., cells or blebs) and to kill any potential cellular contaminants. Chloroform is a volatile organic and is to be used only in a well-ventilated fume hood; discard material containing chloroform as organic waste.
9. Determine the volume recovered after the first chloroform treatment and treat the sample again with an amount of chloroform equal to 10% of that volume. Mix well and centrifuge at 8,000 x g for 10 min. Remove the aqueous (top) layer, being careful to avoid any of the interface or organic layer. Transfer the aqueous layer to a clean tube.
10. Use the phage immediately (as described in step 6) or store at 4 °C.
    ​NOTE: Freezing of φBB-1 phage samples is not recommended. Although the stability of φBB-1 at 4 °C has not been rigorously investigated, samples stored at 4 °C for up to 1 month after recovery have been used successfully in transduction assays.

6. Transduction assay following PEG precipitation of φBB-1 (Figure 1B)

1. Prepare B. burgdorferi cultures to be used as the recipient in transduction assays, as described for the donor strain in step 1 above. Based on the density determined as in step 2, calculate the volume of culture of recipient needed to yield 15 mL of 1 × 10⁷ spirochetes·mL⁻¹.
2. Centrifuge the volume of culture calculated in step 6.1 at 6,000 x g for 10 min. Decant the supernatant and resuspend the pellet in 14.5 mL of fresh BSK.
3. Add ≤500 µL of PEG-precipitated phage sample (from step 5) to the culture of the recipient clone. Mix well and incubate at 33 °C for 72-96 h.
   ​NOTE: The amount of phage recovered during PEG precipitation can be variable, depending on a number of factors. However, from 15 mL culture of induced B. burgdorferi strain CA-11.2A, 500 µL typically contains 50-1,000 viable phage²⁸. Volumes of phage recovery ≥500 µL adversely affect B. burgdorferi growth, likely due to the increased ratio of SM to BSK.
4. Perform the selection of transductants by solid-phase plating after mixing with PEG-precipitated phage as described in step 7.

7. Selection of transductants

NOTE: Solid-phase plating of potential transductants is performed using a single-layer modification of the protocol first described by Samuels¹⁵. B. burgdorferi colonies grow within the agar, so for the selection of transductants by solid-phase plating, the samples must be added to the media while the plates are poured. An alternative method for the selection of transformants using a dilution method in 96-well plates also has been described previously³⁵. This technique also might be effective for the selection of transductants but has not yet been tried for this purpose.

1. Determine the number of plates needed for the selection of transductants based on the number of samples from the transduction assays and controls to be plated.
   NOTE: Typically, two plates are poured per sample, one equivalent to approximately 10% of the culture volume and one that includes the remainder of the culture. Additionally, pour plates that serve as negative controls to individually test that the phage preparation and/or parent clones used as the donor and the recipient do not grow in the presence of the antibiotic(s) used during selection. Preparing plating material for at least two extra plates is also recommended. For example, if the number of samples and controls to be plated is eight, prepare enough plating mix for 10 plates.
2. Prepare solutions for plating as described below.
   NOTE: Each plate will be 30 mL, consisting of 20 mL of 1.5x BSK for plating and 10 mL of 2.1% agarose (2:1 ratio). This will yield a plate with final concentrations of 1x BSK and 0.7% agarose. For example, for 10 plates, prepare a total plating mix of 300 mL, consisting of 200 mL of 1.5x BSK and 100 mL of 2.1% agarose.
   1. Prepare 1 L of 1.5x BSK as described for 1x BSK in step 1.1 using the amounts of each component listed for 1.5x BSK in Table 1. Store as described for 1x BSK.
   2. Prepare 2.1% agarose in water and autoclave. Use the agarose solution fresh or store at room temperature. If stored at room temperature, microwave with the lid on loosely until completely molten prior to plating.
   3. Determine the volume of antibiotic(s) needed to achieve the appropriate concentration (Table 2) based on the entire plating mix.
      NOTE: If the total volume of the final plating solution is 300 mL, mix 200 mL of 1.5x BSK and enough antibiotic to ensure that the entire 300 mL has the correct final antibiotic concentration. If both the donor and the recipient in the transduction assay have different antibiotic-resistance genes, the plating mix should contain both antibiotics. If the PEG-precipitated phage from the donor (as prepared in step 5) encodes an antibiotic-resistance marker and the recipient has none, the plating mix should contain only the one antibiotic.
3. Based on the number of plates to be poured, transfer the appropriate amount of 1.5x BSK and antibiotic(s) to a sterile bottle large enough to hold the entire plating mix. Equilibrate in a water bath at 56 °C for ≥15 min.
4. Equilibrate molten agarose from the autoclave or microwave in a 56 °C water bath for ≥15 min.
5. After equilibration, add the determined amount of 2.1% agarose to the bottle with the 1.5x BSK (with antibiotic) and put the plating solution back in the water bath at 42 °C for 10-15 min.
   NOTE: Higher temperatures can damage or kill the spirochetes³⁶. If using the same water bath as above, cool the water bath to 42-45 °C before starting the timer for the equilibration. Do not let the plating solution equilibrate at 42 °C for more than 20 min or it will start to solidify while pouring the plates.
6. During equilibration, prepare the B. burgdorferi samples to be plated. Transfer the amount to be plated to a sterile 50 mL conical centrifuge tube (see Table of Materials).
   1. If plating an amount less than 1.5 mL (<5% of the final 30 mL plate volume), transfer the sample to the new tube and add the plating mix directly to the sample during plating.
   2. For larger volumes, transfer the desired volume of culture to the new tube and then centrifuge at 6,000 x g for 10 min at room temperature. Decant all but 100-500 µL of the supernatant and use the remainder to resuspend the pellet completely prior to plating.
   3. For control plates following co-culture, add ≥10⁷ cells of the donor or recipient clones to sterile 50 mL conical centrifuge tubes. If the volume is over 1.5 mL, centrifuge and resuspend the pellet as in step 7.6.2. If a transduction assay was performed using the PEG-precipitated phage, in addition to the recipient clone, add 100-250 µL of the phage sample into a sterile 50 mL conical tube for plating.
7. After the plating solution has equilibrated at 42-45 °C for 10-15 min, transfer 30 mL of the plating solution into a tube with the appropriate sample; immediately dispense the plating mix and sample into a labeled plate. Repeat with a fresh pipette for each sample to be plated.
8. Allow the plates to solidify for 15-20 min and then place them in a 33 °C incubator supplemented with 5% CO₂. Do not invert the plates for at least 48 h after being poured.
9. Depending on the background of the recipient clone, check that the colonies appear within the agarose on the selection plates after 10-21 days of incubation. Pick at least 5-10 colonies that grow on the plate in the presence of both antibiotics using a sterilized cotton-plugged 5.75 in borosilicate pipette (see Table of Materials) and inoculate them into 1.5 mL of 1x BSK with the appropriate antibiotic(s).
10. Grow the inoculated colonies at 33 °C as in step 1.3 for 3-5 days or until they reach a density of approximately 20-40 spirochetes per field at 200x magnification using darkfield microscopy.
    ​NOTE: Once screened (see step 8), the spirochetes can be frozen for long-term storage at −80 °C by mixing an equal volume of culture with a mixture of 60% glycerol and 40% 1x BSK sterilized by filtration through a 0.22 µm filter.

8. Verification of potential transductants

NOTE: Screen the clones that grow on plates in the presence of two antibiotics to verify that they represent true transductants in the anticipated (recipient) background. These methods are based on the amplification, and potentially sequencing, of specific regions by the polymerase chain reaction. Detailed protocols and practices of performing PCR in B. burgdorferi are described elsewhere (for a recent example, see Seshu et al.³⁷). Select the primers used for screening the transductants based on the strains used. Some suggestions as to how to approach screening the transductants are described below.

1. Prepare B. burgdorferi lysates for PCR screening.
   NOTE: The following protocol is used to produce washed B. burgdorferi lysates from cultured cells grown as in step 7.9 for immediate analysis of the DNA by PCR. This method is designed to minimize the interference of potential inhibitors in BSK but is not recommended for producing high-quality DNA for sequencing or storage. For that purpose, use a protocol or kit for total genome extraction (see Table of Materials). It is highly recommended that lysates from the parent clones (both donor and recipient strains) also be prepared at the same time to include in each analysis.
   1. Transfer 500 µL of each potential transductant selected and cultured as in step 7.10 to a clean microcentrifuge tube.
   2. Centrifuge the cultures for 10 min at 8,000 x g at room temperature.
   3. Remove the supernatant and resuspend each pellet in 500 µL of TE (10 mM Tris Cl, pH 8.0; 1 mM EDTA, pH 8.0). Centrifuge for 5 min at 8,000 x g at room temperature.
   4. Remove the supernatant and resuspend each pellet in 50 µL of PCR-quality water. Boil the samples for 10 min. Let them cool briefly, and then centrifuge at 8,000 x g for 10 min at room temperature.
   5. For each PCR, immediately use 2 µL from the top of the centrifuged sample; avoid disturbing the pellet.
2. Screen the potential transductants for the specific genes encoding antibiotic resistance using PCR. See Table 4 for the primers for screening antibiotic-resistance markers commonly used in transduction assays.
   NOTE: Although rare in our experience, spontaneous mutation to the aminoglycoside antibiotics used for the selection of heterologous DNA in B. burgdorferi can occur.
3. Screen the potential transductants also using another strain or clone marker to confirm that the background is that of the recipient. Include both the donor and the recipient parent clones in these analyses. Screen for strain-specific markers using sequences based on the strains used and individual laboratory protocols for determining strain integrity.
4. If attempting to transduce into virulent clones for use within the tick vector or mammalian host or for direct comparison with another clone, determine the complete plasmid content of the transductants and the parent clones. This is done to ensure the same plasmid content as that of the comparative strain or the presence of the genetic elements necessary for propagation within the enzootic cycle, as is described elsewhere ^{18,19,37,38,39}.