Bacteriophage-Mediated Gene Delivery in Spirochete Bacteria

Abstract

Source: Eggers, C. H. Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi. J. Vis. Exp. (2022).

This video demonstrates the transduction of genetically modified Borrelia spirochetes using polyethylene glycol–precipitated, non-infectious bacteriophages carrying a kanamycin-resistance plasmid. After incubation with the bacteria, the culture is plated on antibiotic-supplemented medium. Only cells that receive the plasmid survive and form colonies. These colonies are then selected and expanded in antibiotic-containing liquid medium to establish a stable, plasmid-carrying bacterial population for downstream applications

Protocol

1.Transduction assay following PEG (polyethylene glycol) precipitation of bacteriophage φBB-1

1. Prepare B. burgdorferi cultures to be used as the recipient in transduction assays. Based on the density determined, calculate the volume of culture of the recipient needed to yield 15 mL of 1 × 107 spirochetes·mL−1.
2. Centrifuge the volume of culture at 6,000 x g for 10 min. Decant the supernatant and resuspend the pellet in 14.5 mL of fresh BSK (Barbour-Stoenner-Kelly).
3. Add ≤500 µL of PEG-precipitated phage sample 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 a 15 mL culture of the 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 (Suspension Medium) to BSK.
4. Perform the selection of transductants by solid-phase plating after mixing with PEG-precipitated phage.

2. Selection of transductants

NOTE: Solid-phase plating of potential transductants is performed using a single-layer modification of the protocol. 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. 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 using the amounts of each component listed for 1.5x BSK. 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 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 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
7. 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.
   1. 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.
   2. 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. 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.
8. 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.
9. 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.
10. 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 and inoculate them into 1.5 mL of 1x BSK with the appropriate antibiotic(s).
11. Grow the inoculated colonies at 33 °C 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, 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tubes (polypropylene)	USA Scientific	5618-8271
Mitomycin C	Thermo Fisher Scientific	BP25312	CAUTION: potential carcinogen; use only in a chemical fume hood
5.75" Pasteur Pipettes (cotton-plugged/borosilicate glass/non-sterile)	Thermo Fisher Scientific	13-678-8A	Autoclave prior to use
50 mL conical centrifuge tubes (polypropylene)	USA Scientific	1500-1211
Absolute ethanol
Erythromycin	Research Products International Corp	E57000-25.0
Gentamicin reagent solution	Gibco	15750-060
Glucose (Dextrose Anhydrous)	Thermo Fisher Scientific	BP350-500
HEPES	Thermo Fisher Scientific	BP310-500
Kanamycin sulfate	Thermo Fisher Scientific	25389-94-0