Preparation and Induction of Borrelia burgdorferi Cultures for Viral Particle Production

Abstract

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

This video demonstrates the preparation of a genetically modified Borrelia burgdorferi culture engineered with dormant bacteriophage DNA carrying an antibiotic resistance gene. Following antibiotic selection and incubation, a chemical inducer triggers DNA damage, leading to excision, replication, and assembly of new virions containing the resistance gene. After a second centrifugation and resuspension, the bacterial cells harboring mature viral particles are ready for downstream applications.

Protocol

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). 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 a 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 Centers for Disease Control and Prevention (CDC) guidelines.
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.

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 headspace.
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.

Table 1: BSK for the cultivation and selection of B. burgdorferi clones for transduction assays. The formulation and preparation of 1x BSK for culturing B. burgdorferi and 1.5x BSK for the solid-phase selection of B. burgdorferi clones described here are based upon Samuels. Different formulations of BSK (or MKP) that support the growth of B. burgdorferi have not yet been tested using the transduction assay.

Component	1x BSK (for culturing) (1 L)	1.5x BSK (for plating) (1 L)
Bovine serum albumin (fraction V)	35 g	52.5 g
10x CMRL-1066 (without L-glutamine)	8 g	12 g
Neopeptone	4 g	6 g
yeastolate	1.6 g	2.4 g
HEPES	4.8 g	7.2 g
Glucose	4 g	6 g
Sodium citrate	0.56 g	0.84 g
Sodium pyruvate	0.64 g	0.96 g
N-acetyl-glucosamine	0.32 g	0.48 g
Sodium bicarbonate	1.76 g	2.64 g
Heat-inactivated normal rabbit serum (INRS)	66 mL	99 mL

Table 2: 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.

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 3: Potential inducing agents and concentrations used for the induction of φBB-1 from B. burgdorferi. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and ethanol, as well as methanol and isopropanol, have all been demonstrated to induce φBB-1 above constitutive levels in B. burgdorferi strain CA.11-2A. MNNG is a suspected carcinogen and environmental hazard; use with caution, dissolve in a combustible solvent, and incinerate for disposal. Mitomycin C is a suspected carcinogen and potential environmental hazard; use with caution under a chemical fume hood and dispose of properly. While published data suggest that MNNG is the most effective agent for inducing φBB-1, the hazards of working with this chemical and difficulties in acquiring it make its use complicated, particularly with students. Induction with ethanol is consistently better than with methanol or isopropanol and has been most commonly used in the transduction assay.

Inducing agent	Stock concentration (solvent)	Final concentration in the sample
Ethanol	100% (none)	5%
Mitomycin C	2 mg.mL-1 (water)	20 μg.mL-1
1-methyl-3-nitroso-nitroguanidine (MNNG)	50 mg.mL-1 (DMSO)	10 μg.mL-1

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 L filter units (PES, 0.22 µm pore size)	Millipore Sigma	S2GPU10RE
12 mm x 75 mm tube (dual position cap) (polypropylene)	USA Scientific	1450-0810	holds 4 mL with low void volume (for induction)
15 mL conical centrifuge tubes (polypropylene)	USA Scientific	5618-8271
1-methyl-3-nitroso-nitroguanidine (MNNG)	Millipore Sigma		CAUTION: potential carcinogen; no longer readily available, have not tested offered substitute
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
Bacto Neopeptone	Gibco	DF0119-17-9
Bacto TC Yeastolate	Gibco	255772
Bovine serum albumin (serum replacement grade)	Gemini Bio-Products	700-104P
CMRL-1066 w/o L-Glutamine (powder)	US Biological	C5900-01	cell culture grade
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
Millex-GS (0.22 µM pore size)	Millipore Sigma	SLGSM33SS	to filter sterilize antibiotics and other small volume solutions
Mitomycin C	Thermo Fisher Scientific	BP25312	CAUTION: potential carcinogen; use only in a chemical fume hood
N-acetyl-D-glucosamine	MP Biomedicals, LLC	100068
Petri Dish (100 mm × 15 mm)	Thermo Fisher Scientific	FB0875712
Rabbit serum non-sterile trace-hemolyzed young (NRS)	Pel-Freez Biologicals	31119-3	heat inactivate as per manufacturer's instructions
Sodium bicarbonate	Thermo Fisher Scientific	BP328-500
Sodium pyruvate	Millipore Sigma	P8674-25G
Streptomycin sulfate solution	Millipore Sigma	S6501-50G
Trisodium citrate dihydrate	Millipore Sigma	S1804-500G	sodium citrate for BSK