Phage Purification from Borrelia burgdorferi

Streszczenie

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

This video demonstrates the isolation of borrelial phage particles from a Borrelia burgdorferi suspension using polyethylene glycol-mediated phase separation. The sample is centrifuged, treated with a salt solution, and a molecular crowding agent to precipitate phages. Chloroform extraction is done to remove debris and concentrate phage particles. The purified phage is stored under cold conditions for further applications.

Protokół

1. Polyethylene glycol (PEG) precipitation to recover phage

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 B. burgdorferi culture infected with phage with the appropriate antibiotic at the concentration indicated in Table 1. Incubate the sample at 33 °C for 72-96 h.
2. Prepare solutions for PEG precipitation.
   1. Prepare 500 mL of 5 M sodium chloride (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 magnesium sulfate (MgSO₄), and 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) [pH 7.5]). Sterilize by autoclaving. Store at 4 °C.
3. For PEG precipitation of phage from the donor B. burgdorferi clone, after 72-96 h of incubation with phage, 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 the original supernatant, but depending on the pellet size, 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 treatment. 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 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.

Antibiotic	Stock concentration	Final concentration in the culture of Bb
Kanamycin	100 mg.mL-1 (in water)	200-400 μg.mL-1
Gentamicin	50 mg.mL-1 (in water)	50 μg.mL-1
Streptomycin	50 mg.mL-1 (in water)	50 μg.mL-1
Erythromycin	2 mg.mL-1 (in EtOH)	0.06 μg.mL-1

Table 1: Potential antibiotics and concentrations to be used for the selection and maintenance of heterologous DNA in B. burgdorferi. This list is based on current antibiotic-resistant markers commonly used in Borrelia burgdorferi. Kanamycin, gentamicin, and streptomycin are prepared in water, filter-sterilized through a 0.22 µM filter, and stored at −20 °C. Erythromycin is prepared in 95% ethanol and stored at −20 °C. Many laboratories report the successful use of kanamycin for selection at a concentration of 200 µg·mL⁻¹; when using both gentamicin and kanamycin for selection in transduction assays, 400 µg·mL⁻¹ kanamycin is used. The aadA gene confers resistance to both streptomycin and spectinomycin. For the selection of constructs containing the aadA gene in E. coli, 100 µg·mL⁻¹ spectinomycin is used. Note that the aminoglycosides (kanamycin, gentamicin, and streptomycin) are not clinically relevant in the treatment of Lyme disease; however, erythromycin is used clinically in certain situations. Although natural resistance to this antibiotic in B. burgdorferi has been reported, this resistance marker has not been used thus far in the transduction assays reported here.

Materiały

Lista materiałów użytych w tym artykule

Nazwa	Firma	Numer katalogowy	Komentarze
15 mL Conical centrifuge tubes (polypropylene)	USA Scientific	5618-8271
50 mL Conical centrifuge tubes (polypropylene)	USA Scientific	1500-1211
Absolute ethanol
Chloroform (for molecular biology)	Thermo Fisher Scientific	BP1145-1	CAUTION: volatile organic; use only in a chemical fume hood
Polyethylene glycol 8000 (PEG)	Thermo Fisher Scientific	BP233-1
Sodium chloride	Thermo Fisher Scientific	BP358-1
Trisodium citrate dihydrate	Millipore Sigma	S1804-500G	sodium citrate for BSK