Isolation of Bacteriophages from Environmental Sources, and Creation and Functional Screening of Phage DNA Libraries

Adam J. Pelzek1, Raymond Schuch2, Jonathan E. Schmitz3, Vincent A. Fischetti4

1 New York University School of Medicine, New York, New York, 2 ContraFect Corporation, Yonkers, New York, 3 Vanderbilt University Medical Center, Nashville, Tennessee, 4 The Rockefeller University, New York, New York
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 13.3
DOI:  10.1002/9780470089941.et1303s07
Online Posting Date:  October, 2013
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Abstract

Bacteriophages (phages) have evolved specific classes of proteins such as adhesins and lysins to mediate specific bacterial host recognition and rapid and efficient lysis following infection. However, because many bacterial species, and thus phages, cannot be cultured in the laboratory, techniques for the efficient cloning and screening of phage gene libraries are required to aid in the discovery of novel phage proteins for use in diagnostic and therapeutic applications. This article contains protocols for the isolation of phages from environmental samples, enrichment of phages targeting host bacteria of interest, and induction of phages from lysogenized host strains. We also provide an optimized protocol for the creation and functional screening of phage DNA libraries derived from environmental samples. Curr. Protoc. Essential Lab. Tech. 7:13.3.1‐13.3.35. © 2013 by John Wiley & Sons, Inc.

Keywords: bacteriophage; phage; phage isolation; phage culture; metagenomics; phage titer; phage characterization; phage DNA; single‐step growth curve; DNA library; lysin screen

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning (Host‐Bacterium Considerations)
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Isolation of Bacteriophages from Environmental Samples
  • Basic Protocol 2: Creation of Bacterial Lysogens
  • Basic Protocol 3: Isolation of Prophages Induced from Lysogenized Host Strains
  • Basic Protocol 4: Concentration of Phage Particles and Buffer Replacement
  • Basic Protocol 5: Phage DNA Isolation
  • Alternate Protocol 1: Phage DNA Isolation with a Qiagen Lambda Kit
  • Basic Protocol 6: Phage DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis (PFGE)
  • Basic Protocol 7: Creation and Screening of Phage DNA Libraries from Genomic, Multigenomic, and Metagenomic Sources
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Bacteriophages from Environmental Samples

  Materials
  • Environmental source material (soil, sand, water, feces, food, etc.)
  • Phosphate‐buffered saline (PBS; unit 3.3)
  • Bacterial host strain (see unit 4.4) and appropriate growth medium
  • Soft (top) agar and hard (bottom) agar (see unit 4.4, Basic Protocol 1)
  • 2× concentrated growth medium appropriate for bacterial host
  • Chloroform (optional)
  • Erlenmeyer flasks (autoclaved, size relative to the amount of environmental material to be processed)
  • Cheesecloth (double layers, wrapped in an aluminum foil packet and autoclaved)
  • 0.22‐µm sterile filtration apparatuses for bottle tops (Millipore,150 and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck‐size for your bottles)
  • 13‐ml round‐bottom polypropylene tubes, sterile (Sarstedt)
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for isolation, culture, and characterization of bacteriophages (unit 4.4), concentration of phage particles ( protocol 4), and isolation of phage‐cured bacterial derivatives ( protocol 3)

Basic Protocol 2: Creation of Bacterial Lysogens

  Materials
  • High‐titer culture of bacteriophage (unit 4.4)
  • QIAprep spin miniprep kit (Qiagen; alternatively see protocol 5)
  • Phage‐specific PCR primers (order from oligo synthesis service, e.g., Fisher Scientific)
  • Bacterial host strain (see unit 4.4) and appropriate growth medium
  • LB medium (unit 4.2) containing 15% (v/v) glycerol
  • Brain‐Heart Infusion (BHI) broth and plates (BD Difco; BHI medium is typically used for Bacillus strains)
  • 0.5 M NaOH
  • 1 M Tris·Cl, pH 8.0 (unit 3.3)
  • PCR master mix (e.g., Qiagen Taq Polymerase PCR Master Mix kit; see unit 10.2)
  • 1 Kb Plus DNA Ladder (Invitrogen)
  • Mitomycin C (DNA‐damaging agent, handle with gloves/mask)
  • Non‐lysogenized reporter strain of bacterial host
  • 30°C shaking water bath
  • 30°C bacterial incubator
  • PCR tubes
  • Thermal cycler for PCR reactions
  • 0.22‐µm sterile filtration apparatuses for bottle tops (Millipore,150 and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck‐size for your bottles)
  • Additional reagents and equipment for PEG precipitation of bacteriophage ( protocol 4) and DNA isolation ( protocol 5 or Alternate Protocol) from bacteriophages, DNA library production ( protocol 8), colony PCR (unit 4.2), BLAST searching (unit 11.1), isolation, culture, and characterization of bacteriophages (unit 4.4), and the polymerase chain reaction (PCR; unit 10.2)

Basic Protocol 3: Isolation of Prophages Induced from Lysogenized Host Strains

  Materials
  • Bacterial strain lysogenized with phage and appropriate culture medium
  • Bacterial reporter strain (non‐lysogenized; different from the lysogenized host strain)
  • Buffer: PBS (unit 3.3), normal saline (0.9% w/v NaCl), or 0.1 M MgSO 4
  • Phage titering diluent: culture medium, PBS (unit 3.3) or 5 mM CaCl 2
  • Mitomycin C (DNA‐damaging agent, handle with gloves/mask)
  • 0.1 M MgSO 4
  • LB medium (unit 4.2)
  • Spectrophotometer for determining OD 600
  • UV light (254‐nm) source
  • UV face protection screen (Carl Roth GmbH & Co., cat. no. CA34.1)
  • Petri dishes, sterile
  • Bacterial incubator
  • Tabletop centrifuge and microcentrifuge
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Additional reagents and equipment for titering bacteria ( protocol 1) and phage (unit 4.4)

Basic Protocol 4: Concentration of Phage Particles and Buffer Replacement

  Materials
  • Phage‐containing supernatant from freshly grown bacterial culture (from protocol 3 in this unit or unit 4.4, Basic Protocol 3)
  • Culture medium
  • Buffer: e.g., phosphate buffered saline (PBS; unit 3.3), pH 7.4
  • 1 M NaCl
  • Polyethylene glycol 8000 (PEG 8000, powder)
  • Phage buffer (see recipe)
  • 0.1 M MgSO 4
  • 0.22‐µm sterile filtration apparatuses for bottle tops (Millipore,150 and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck‐size for your bottles)
  • Stir Cell 8400 (400‐ml size, Millipore, autoclaved to sterilize; check with manufacturer for handling, and autoclave each component of the stir cell separately in aluminum foil); pressurized from CO 2 tank
  • Cellulose ultrafiltration filters (Millipore, 10‐kDa or 100‐kDa cutoff)
  • Centrifuge bottles: 500‐ml for GS‐3 rotor; wash, wrap with lid (not sealed) in aluminum foil, and autoclave
  • Oak Ridge polypropylene centrifuge tubes (∼30 ml; compatible with Sorvall SS‐34 rotor)
  • Centrifuge (floor model, Beckman or Sorvall) with angle rotor GS‐3, GSA, or SS‐34
  • End‐over‐end rotator
  • Centrifuge (ultracentrifuge, Beckman) with >30,000 rpm rotor and Ultra Clear tubes
  • Additional reagents and equipment for titering bacteriophage ( protocol 1)

Basic Protocol 5: Phage DNA Isolation

  Materials
  • Phage lysate ( protocol 1)
  • TE buffer, pH 8.0 (unit 3.3)
  • Lambda buffer: 10 mM Tris·Cl, pH 7.4 (unit 3.3) plus 10 mM MgCl 2
  • DNase I (create a 1 mg/ml stock, then add to lysate for 1 µg/ml final concentration)
  • RNAse A (create a 12.5 mg/ml stock, then add to lysate for 10 µg/ml final concentration)
  • Polyethylene glycol 8000 (PEG 8000, powder)
  • 25:24:1 phenol:chloroform:isoamyl alcohol (unit 5.2)
  • 3 M sodium acetate (unit 3.3)
  • 95% and 70% ethanol (ultrapure, lab grade)
  • 0.22‐µm sterile filtration apparatuses for bottle tops (Millipore,150 and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck‐size for your bottles)
  • NanoDrop spectrophotometer (optional; can measure DNA concentrations in 1‐ to 2‐µl aliquots; also see unit 2.2)
  • Qiagen DNeasy Blood & Tissue kit (optional)
  • Additional reagents and equipment for titration of phages ( protocol 1), PEG precipitation of phage particles ( protocol 4), purification and concentration of nucleic acids (unit 5.2), and quantification of nucleic acids by Nanodrop (unit 2.2)

Alternate Protocol 1: Phage DNA Isolation with a Qiagen Lambda Kit

  Materials
  • Phage lysate ( protocol 1)
  • Buffers included in Qiagen Lambda kit:
    • L1 buffer: 300 mM NaCl; 100 mM Tris·Cl, pH 7.5 (unit 3.3); 10 mM EDTA; 0.2 mg/ml BSA; 20 mg/ml RNase A; 6 mg/ml DNase I (store 4°C)
    • L2 buffer: 30% polyethylene glycol (PEG 6000); 3 M NaCl (chill to 4°C before use)
    • L3 buffer: 100 mM NaCl; 100 mM Tris·Cl, pH 7.5 (unit 3.3); 25 mM EDTA (store at room temperature)
    • L4 buffer: 4% sodium dodecyl sulfate (SDS), store at room temperature (check for SDS precipitation before use, warm at 37°C if necessary)
    • L5 buffer: 3 M potassium acetate, pH 5.5 (store at room temperature)
    • QBT buffer (column equilibration buffer): 750 mM NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol; 0.15% Triton X‐100 (store and room temperature)
    • QC buffer (column wash buffer): 1.0 M NaCl, 50 mM MOPS, pH 7.0; 15% isopropanol (store at room temperature)
    • QF buffer (column elution buffer): 1.25 M NaCl; 50 mM Tris·Cl, pH 8.5 (unit 3.3); 15% (v/v) isopropanol (store at room temperature)
  • Isopropanol
  • 70% ethanol (ultrapure, lab‐grade)
  • TE buffer (unit 3.3)
  • Qiagen tip DNA column: Qiagen tip‐100 (midi‐prep) or Qiagen tip‐500 (Maxi‐prep)
  • 70°C water bath
  • 0.22‐µm sterile filtration apparatuses for bottle tops (Millipore,150 and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck‐size for your bottles)
  • Oak Ridge polypropylene centrifuge tubes (∼30 ml; compatible with Sorvall SS‐34 rotor)
  • Additional reagents and equipment for quantification of nucleic acids (unit 2.2)

Basic Protocol 6: Phage DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis (PFGE)

  Materials
  • 0.5× TBE buffer (unit 7.2)
  • Agarose (also see unit 7.2)
  • Milli‐Q water
  • Restriction‐enzyme‐cleaved phage DNA (see introduction to this protocol)
  • DNA PFGE markers (low and mid‐range, New England Biolabs)
  • DNA Ladder (1 KB Plus, Invitrogen)
  • 0.5 µg/ml ethidium bromide in 0.5× TBE buffer (wear gloves, this is a mutagen), or other DNA stain (see unit 7.4)
  • CHEF DR II system apparatus for pulsed‐field gel electrophoresis (BioRad) with refrigeration
  • Gel cast tray, box, and well inserts (also see unit 7.2)
  • Heat block (capable of controlling temperature from 16° to 70°C), for incubation and heat‐killing of restriction enzyme
  • Alpha‐Imager (Cell Biosciences), or similar UV light box with camera for gel picture acquisition
  • Additional reagents and equipment for agarose gel electrophoresis (unit 7.2)
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Figures

Videos

Literature Cited

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Key References
  Clokie, M.R.J. and Kropinski, A.M. 2008. Bacteriophages: Methods and Protocols, Volume 2: Humana Press, Totowa, N.J.
  For molecular biology protocols concerning phage characterization.
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2013. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Resource for general molecular biology methods.
  Adams, M.H. 1959. Bacteriophages. Interscience Publishers, New York. Available at http://www.archive.org/details/bacteriophages00adam.
  Mark Adams was a central figure in the field of bacteriophage research. His text Bacteriophages integrated basic phage knowledge with various laboratory techniques. It is currently available free online at the Biodiversity Heritage Library Internet Archive (http://www.archive.org/details/bacteriophages00adam).
  Cairns, J., Stent, G.S., and Watson, J.D. 2007. Phage and the Origins of Molecular Biology (Expanded Edition). 2007. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Another influential reference, first published in 1966.
  Calendar, R.L. and Abedon, S.T. 2005. The Bacteriophages, Second Edition. Oxford University Press, Oxford.
  More recent influential references on bacteriophages.
  Waldor, M.K., Freidman, D.I., and Adhya, S.L. 2005. Phages: Their Role in Bacterial Pathogenesis and Biotechnology, First Edition. American Society for Microbiology Press, Washington, D.C.
  A primary resource for this article, and a very valuable reference, which contains a detailed compendium on the history of phage research, as well as an extensive appendix of techniques.
  Kutter, E. and Sulakvelidze, A. 2005. Bacteriophages: Biology and Applications. CRC Press, Boca Raton, Fla.
  A book that focuses on the development of bacteriophage lysins and other phage therapy‐based strategies.
  Villa, T.G. and Veiga‐Crespo. 2010. Enzybiotics: Antibiotic Enzymes as Drugs and Therapeutics. pp. 199‐218. John Wiley & Sons, Hoboken, N.J.
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