Genetic Manipulation of Mycobacterium abscessus

Halima Medjahed1, Anil Kumar Singh1

1 Inserm U 1002, Unité de Pathogénie des Infections Systémiques, Paris, France
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10D.2
DOI:  10.1002/9780471729259.mc10d02s18
Online Posting Date:  August, 2010
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Abstract

This unit covers genetic manipulation protocols for M. abscessus, including nucleic acid extraction (plasmid DNA, genomic DNA, and RNA), transformation, and a recombineering mutagenesis method. M. abscessus is a Biosafety Level 2 (BSL‐2) bacterium, and working considerations are also discussed. Curr. Protoc. Microbiol. 18:10D.2.1‐10D.2.19. © 2010 by John Wiley & Sons, Inc.

Keywords: chronic lung disease; cystic fibrosis; genomic DNA; plasmid; RNA; transformation; recombineering mutagenesis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Extraction of Genomic DNA by Using Lysozyme Method
  • Alternate Protocol 1: Extraction of Genomic DNA Using the BeadBeater‐Phenol Method
  • Alternate Protocol 2: Small‐Scale, Rapid Extraction of Genomic Mycobacterium abscessus DNA for PCR
  • Basic Protocol 2: Extraction of Plasmid DNA
  • Basic Protocol 3: Extraction of RNA with TRIzol
  • Basic Protocol 4: Electrotransformation of M. abscessus
  • Basic Protocol 5: Mutagenesis of M. abscessus by Recombineering
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction of Genomic DNA by Using Lysozyme Method

  Materials
  • M. abscessus culture and appropriate medium (unit 10.1)
  • Lysis buffer I (see recipe)
  • Lysis buffer II (see recipe)
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol (Sigma, cat. no. P3803; also see appendix 2A)
  • 5 M NaCl
  • Absolute ethanol, ice cold
  • 70% ethanol
  • TE buffer (see recipe)
  • Spectrophotometer
  • 15‐ml polypropylene conical tubes
  • Tabletop centrifuge
  • 2‐ml microcentrifuge tubes
  • Heating blocks
  • Additional reagents and equipment for growing M. abscessus (unit 10.1)

Alternate Protocol 1: Extraction of Genomic DNA Using the BeadBeater‐Phenol Method

  • Sucrose Lysis Solution I (see recipe)
  • Sucrose Lysis Solution II (see recipe)
  • Proteinase K (see recipe)
  • Isopropanol
  • 0.1 mm silica/zirconium beads (BioSpec cat. no. 11079101z)
  • FastPrep machine (BioSpec, cat. no. 607)
  • Lysis Matrix B tube (MP Biomedical, cat. no. 6911‐100; http://www.mpbio.com)
  • Heating block

Alternate Protocol 2: Small‐Scale, Rapid Extraction of Genomic Mycobacterium abscessus DNA for PCR

  Materials
  • Plated colonies or liquid cultures of M. abscessus (unit 10.1)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M ammonium acetate ( appendix 2A)
  • 95% ethanol
  • Plastic inoculating loop
  • 500‐µl microcentrifuge tubes with screw caps and O‐rings
  • Heat block

Basic Protocol 2: Extraction of Plasmid DNA

  Materials
  • M. abscessus cell culture and appropriate medium (unit 10.1)
  • Lysozyme solution (see recipe) for supplementing Buffer P1 from QIAprep kit
  • 50‐ml conical centrifuge tubes
  • Tabletop centrifuge
  • QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27106)
  • 2‐ml microcentrifuge tubes
  • Additional reagents and equipment for growing M. abscessus (unit 10.1)

Basic Protocol 3: Extraction of RNA with TRIzol

  Materials
  • M. abscessus cell culture and appropriate medium (unit 10.1)
  • RNAprotect Bacteria Reagent (Qiagen, cat. no.76506)
  • TRIzol (Invitrogen, cat. no. 15596)
  • Chloroform
  • Acid phenol (Invitrogen, cat. no. AM9720)
  • Isopropanol
  • 75% ethanol (made with DEPC‐treated H 2O)
  • DEPC‐treated H 2O ( appendix 2A)
  • RNase OUT (Invitrogen, cat. no. 10777‐019)
  • RNeasy Mini Kit (Qiagen, cat. no. 74104)
  • 1% agarose gel (Voytas, )
  • Refrigerated centrifuge
  • Lysis Matrix B tube (MP Bio, cat. no. 6911‐100; http://www.mpbio.com)
  • 2‐ml microcentrifuge tubes
  • Fast RNA Prep machine (FP120, Bio101, Thermo Electron Corporation).
  • Additional reagents and equipment for growing M. abscessus (unit 10.1) and agarose gel electrophoresis (Voytas, )

Basic Protocol 4: Electrotransformation of M. abscessus

  Materials
  • M. abscessus cell culture (unit 10.1)
  • Luria‐Bertani (LB) medium or Middlebrook 7H9 broth (unit 1.1)
  • 10% glycerol (see recipe)
  • Sterile DNA of interest
  • Selective plates
  • 500‐ml Erlenmeyer or conical flask (Borosil, cat no. 4980)
  • Shaking incubator
  • 50‐ml conical centrifuge tubes
  • Tabletop centrifuge
  • Electroporator (e.g., BioRad GenePulser)
  • 0.2‐cm electroporation cuvettes (GenePulser cuvette; BioRad, cat. no. 165‐2086)
  • 15‐ml snap‐cap plastic tube (Falcon cat. no. 2059 or equivalent)
  • Additional reagents and equipment for growing M. abscessus (unit 10.1)
NOTE: Each step of the preparation of electrocompetent cells should be carried out on ice, and all centrifugation steps should be carried out at 4°C.

Basic Protocol 5: Mutagenesis of M. abscessus by Recombineering

  Materials
  • PCR primers for deletion of target DNA sequence
  • Template containing target DNA sequence
  • Necessary restriction enzymes for cloning
  • pBluescriptII SK (+/−) (Stratagene)
  • pLYG‐204 (Gao et al., )
  • Luria‐Bertani agar or Middlebrook 7H10/OADC plate (unit 10.1) with 50 µg/ml zeocin (no kanamycin)
  • PCR purification kit (Qiagen, cat. no. D6943‐02)
  • pJV53 (van Kessel and Hatfull, )
  • M. abscessus cell culture
  • Luria‐Bertani or Middlebrook 7H10/OADC agar plates (unit 10.1) containing 50 µg/ml kanamycin
  • Luria‐Bertani (LB) or Middlebrook 7H9 liquid medium (unit 10.1) containing 50 µg/ml kanamycin
  • 20% succinate (see recipe)
  • 20% acetamide (see recipe)
  • 10% glycerol (see recipe)
  • Luria‐Bertani (LB) or Middlebrook 7H9 liquid medium (unit 10.1)
  • Luria‐Bertani agar or Middlebrook 7H10/OADC plate (unit 10.1) with 50 µg/ml kanamycin and 50 µg/ml zeocin
  • Luria‐Bertani (LB) or Middlebrook 7H9 liquid medium (unit 10.1) containing 50 µg/ml zeocin (no kanamycin)
  • Electroporator (e.g., BioRad Gene Pulser)
  • 0.2‐cm electroporation cuvettes (GenePulser cuvette; BioRad, cat. no. 165‐2086)
  • 500‐ml Erlenmeyer or conical flask (Borosil, cat. no. 4980)
  • Tabletop centrifuge
  • 50‐ml conical tubes
  • Additional reagents and equipment for PCR (Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), and growing M. abscessus (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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