Phage Purification from Borrelia burgdorferi

Abstract

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.

Protocol

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). Steril....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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