Characterization of Bacteria in Mixed Biofilm Communities Using Denaturing Gradient Gel Electrophoresis (DGGE)

Allana K. Welsh1, Robert J.C. McLean1

1 Texas State University‐San Marcos, San Marcos, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.1
DOI:  10.1002/9780471729259.mc01e01s4
Online Posting Date:  February, 2007
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Abstract

Most microorganisms in nature, including those within biofilms, live in mixed populations. PCR‐based molecular genetic techniques are very useful in studying microbial diversity since unculturable as well as culturable organisms can be investigated. One such technique is denaturing gradient gel electrophoresis (DGGE), which separates PCR‐amplified community 16S rRNA (and other gene) sequences on the basis of G+C content. Unlike other community fingerprinting techniques, bands from DGGE gels can be excised, and sequenced to identify community members. Thus DGGE can be used to describe overall microbial diversity as well as to identify individual community members. This protocol describes a method for using DGGE to study microbial diversity within biofilm populations.

Keywords: Biofilms; denaturing gradient gel electrophoresis; DNA fingerprint; 16S rRNA; microbial diversity

     
 
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Table of Contents

  • Basic Protocol 1: Denaturing Gradient Gel Electrophoresis
  • Support Protocol 1: PCR of Biofilm Samples for DGGE
  • Support Protocol 2: Sonication of Field Samples and Extraction of DNA
  • Support Protocol 3: Preparation of DGGE Standards
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Denaturing Gradient Gel Electrophoresis

  Materials
  • 6% acrylamide solutions containing 30% (“LO”) and 50% (“HI”) denaturant (see recipe)
  • 0.5× TAE buffer: mix 100 ml 50× TAE ( appendix 2A) in a final volume of 10 liters H 2O for at least 1 hr; store up to 3 months at room temperature
  • Ammonium persulfate (APS): store up to 1 year at 4°C
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED): store up to 1 year at 4°C
  • PCR‐amplified samples and standards (Support Protocols protocol 21 and protocol 43)
  • 2× DGGE gel loading dye (see recipe)
  • Ethidium bromide staining solution (see recipe)
  • TE buffer ( appendix 2A)
  • DGGE System, e.g., DCode Universal Mutation Detection System (Bio‐Rad) for 16‐cm parallel gels, including:
    • Glass plates with spacers (grooved, 1 mm), two pairs
    • Sandwich clamps
    • Sponges
    • Casting stand
    • Alignment card
    • 30‐ml syringes, syringe sleeves, syringe cap screws, luer syringe locks, and luer couplings
    • Tygon tubing
    • Model 475 gradient former
    • Y fitting
    • 16‐well combs
    • DCode lid stand
    • Electrophoresis/temperature control module
    • Electrophoresis tank
    • Sandwich core
  • Absorbent bench paper
  • 50‐ml conical polypropylene tubes
  • 20‐G needle
  • 250‐ml beakers
  • Micropipettor with gene sequencing tips
  • Power supply (e.g., Bio‐Rad Power Pac 300)
  • Plastic mesh shelf liner (available from many stores, e.g., Wal‐Mart)
  • UV transilluminator
  • Scalpel, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Digital or other camera for gel documentation
  • Additional reagents and equipment for sequencing DNA (e.g., Slatko et al., ; optional)

Support Protocol 1: PCR of Biofilm Samples for DGGE

  Materials
  • AmpliTaq GOLD Polymerase, 5 U/µl (Applied Biosystems)
  • 10× PCR buffer II (Applied Biosystems)
  • 25 mM MgCl 2 (Applied Biosystems)
  • 10 mM dNTP blend (Applied Biosystems)
  • Primers (Table 1.1.1): dilute to 20 µM
  • DNA template: extracted from biofilm sample ( protocol 3)
  • Nuclease‐free PCR water (Applied Biosystems)
  • Low mass DNA ladder (Invitrogen)
  • 100% isopropanol
  • 3 M sodium acetate pH 5.2 ( appendix 2A)
  • 70% ethanol
  • TE buffer ( appendix 2A)
  • 0.2‐ml PCR tubes, sterile
  • Thermocycler with block for 0.2 ml tubes
  • −80°C freezer
  • Vacuum centrifuge (Savant; optional)
  • Additional reagents and equipment for performing agarose gel electrophoresis and ethidium‐bromide staining (see Voytas, )

Support Protocol 2: Sonication of Field Samples and Extraction of DNA

  Materials
  • Biofilm sample (from the field)
  • 95% or 100% ethanol
  • Sterile deionized H 2O
  • R2A agar medium (Difco): prepare agar plates according to manufacturer's instructions
  • TE buffer ( appendix 2A)
  • DNeasy tissue kit (Qiagen)
  • Enzymatic lysis buffer: 20 mM Tris·Cl, pH 8.0 ( appendix 2A)/2 mM EDTA ( appendix 2A)/1.2% Triton X‐100
  • Lysozyme (Fisher Scientific)
  • Writing paper
  • Petri dishes, sterile
  • Scalpel, sterile
  • Glass scintillation vials: Fisher brand 20‐ml borosilicate vials with polypropylene caps recommended, sterile
  • Bath sonicator (e.g., Branson Ultrasonic Cleaner Model 1510MT): fill to a depth of ∼1 cm prior to use; drain and clean after use
  • 50‐ml centrifuge tubes
  • Vacuum filter unit with 0.2‐µm filter

Support Protocol 3: Preparation of DGGE Standards

  • Bacterial strains for use in the standard—e.g., Escherichia coli MG1655 (ATTC #47076), Pseudomonas aeruginosa (ATCC #10145), and Chromobacterium violaceum (ATCC #12472)
  • Appropriate medium for bacteria used ( appendix 2C)
  • DNeasy tissue kit (Qiagen)
CAUTION:E. coli, P. aeruginosa, and C. violaceum are Biosafety Level 2 (BSL‐2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See unit 1.1 and other pertinent resources ( appendix 1B) for more information.
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Figures

Videos

Literature Cited

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   Slatko, B.E., Eckert, R.L., Albright, L.M., and Ausubel, F.M. 1999. DNA sequencing strategies. In Current Protocols in Molecular Biology. (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.) pp. 7.1.1‐7.1.7. John Wiley and Sons, Hoboken, N.J.
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Internet Resources
   http://www1.qiagen.com/literature/handbooks/PDF/GenomicDNAStabilizationAndPurification/FromAnimalAndPlantIssues/DNY_BloodTissue/1037951_HB_DNY_Blood_Tissue_062006_Web.pdf
  The Qiagen DNeasy tissue kit manual gives directions for extraction of DNA from Gram‐positive and Gram‐negative bacteria.
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