Genetic Manipulation of Mycobacterium tuberculosis

Michelle H. Larsen1, Karolin Biermann1, Steven Tandberg1, Tsugunda Hsu1, William R. Jacobs,1

1 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.2
DOI:  10.1002/9780471729259.mc10a02s6
Online Posting Date:  August, 2007
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Abstract

This unit includes protocols for the genetic manipulation of Mycobacterium tuberculosis, including nucleic acid extraction (plasmid DNA, genomic DNA, and mRNA), and methods for electroporation (transformation), transduction (including allelic exchange), and transposon mutagenesis. Considerations for working with M. tuberculosis at Biosafety Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.2.1‐10A.2.21. © 2007 by John Wiley & Sons, Inc.

Keywords: tuberculosis; plasmid; genomic DNA; mRNA; transformation; transduction; bacteriophage; mutagenesis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Extraction of Genomic DNA Using the CTAB‐Lysozyme Method
  • Alternate Protocol 1: Extraction of Genomic DNA Using the GTC Method
  • Alternate Protocol 2: Small‐Scale, Rapid Extraction of Genomic Mycobacterium tuberculosis DNA for PCR
  • Basic Protocol 2: Extraction of Plasmid DNA
  • Basic Protocol 3: Extraction of RNA with Trizol
  • Alternate Protocol 3: Extraction of RNA from Mycobacterium tuberculosis Using the Fast RNA Prep
  • Support Protocol 1: GTC Method for Stabilization of the Mycobacterium tuberculosis mRNA Pool
  • Basic Protocol 4: Electrotransformation of Mycobacterium tuberculosis
  • Basic Protocol 5: Generating Mycobacterium tuberculosis–Knockout Mutants with Specialized Transduction
  • Support Protocol 2: Lambda Phage Transduction of E. coli HB101
  • Support Protocol 3: Electroporation of M. smegmatis mc2155
  • Basic Protocol 6: Transposon Mutagenesis of Mycobacterium tuberculosis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction of Genomic DNA Using the CTAB‐Lysozyme Method

  Materials
  • 10% (w/v) glycine (see recipe)
  • Cell culture
  • GTE solution (see recipe)
  • Lysozyme solution (see recipe)
  • 10% SDS (see recipe)
  • 10 mg/ml proteinase K (see recipe)
  • 5 M NaCl
  • CTAB solution (see recipe), 65°C
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • Isopropanol
  • 70% ethanol
  • TE buffer ( appendix 2A)
  • 37°C incubator
  • 15‐ml polypropylene conical tubes
  • Refrigerated tabletop centrifuge
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge

Alternate Protocol 1: Extraction of Genomic DNA Using the GTC Method

  Materials
  • M. tuberculosis cell culture
  • 3:1 (v/v) chloroform/methanol (using water‐saturated chloroform)
  • Phenol, Tris‐buffered ( appendix 2A)
  • GTC solution (see recipe)
  • 2‐Mercaptoethanol
  • Isopropanol
  • 70% ethanol
  • TE buffer ( appendix 2A)
  • Spectrophotometer
  • 50‐ml polypropylene conical tubes
  • Tabletop centrifuge
  • Vortex

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

  Materials
  • Plated colonies or liquid cultures
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M ammonium acetate
  • 95% ethanol
  • Plastic loop
  • 200 µl microcentrifuge tubes with screw‐caps and O‐rings
  • Vortex
  • 95°C heating blocks
  • Microcentrifuge

Basic Protocol 2: Extraction of Plasmid DNA

  Materials
  • 10% (w/v) glycine (see recipe)
  • Cell culture
  • Qiagen midi‐prep kit containing:
    • P1 buffer
  • 10 mg/ml lysozyme
  • 1:1 (v/v) chloroform/methanol

Basic Protocol 3: Extraction of RNA with Trizol

  Materials
  • M. tuberculosis culture
  • 3:1 (v/v) chloroform/methanol, prepare fresh
  • Trizol
  • Chloroform
  • Isopropanol
  • 70% ethanol (made with DEPC‐treated water), cold
  • Diethylpyrocarbonate (DEPC)‐treated water
  • Spectrophotometer
  • 15‐ml polypropylene tubes
  • Tabletop centrifuge
  • Vortex
  • 13‐ml Sarstedt tube
  • 1.5‐ and 2‐ml RNase‐free tubes (Ambion)

Alternate Protocol 3: Extraction of RNA from Mycobacterium tuberculosis Using the Fast RNA Prep

  Materials
  • Mycobacterial culture
  • Qiagen RNA protect reagent (Qiagen cat. no. 76506)
  • Qiagen RNeasy kit (Qiagen cat. no. 74104)
  • Absolute ethanol
  • Spectrophotometer
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge
  • Lysing Matrix B tube (MP Bio cat. no. 6911‐100)
  • Fast RNA prep machine (FastPrep; FP120, Bio101, Thermo Electron Corporation)

Support Protocol 1: GTC Method for Stabilization of the Mycobacterium tuberculosis mRNA Pool

  Materials
  • M. tuberculosis culture
  • GTC solution (for RNA prep; see recipe)
  • 15‐ or 50‐ml conical tubes
  • Tabletop centrifuge

Basic Protocol 4: Electrotransformation of Mycobacterium tuberculosis

  Materials
  • M. tuberculosis culture
  • Middlebrook 7H9 broth
  • 10% glycerol with Tween‐80 (see recipe), sterile
  • DNA
  • Selective plates
  • Spectrophotometer
  • 490‐cm2 roller bottle (Corning cat. no. 430195)
  • 37°C incubator
  • 50‐ml conical tubes
  • Tabletop centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 0.2‐cm electroporation cuvettes
  • Electroporator (e.g., BioRad Gene Pulser)
  • 15‐ml snap‐cap plastic tube (Falcon, cat. no. 2059 or equivalent)

Basic Protocol 5: Generating Mycobacterium tuberculosis–Knockout Mutants with Specialized Transduction

  Materials
  • Upstream and downstream regions (1‐kb each) of gene of interest
  • pYUB854 plasmid or equivalent ( ; Bardarov et al., )
  • PacI restriction endonuclease and buffer 1
  • phAE159 or equivalent ( )
  • Shrimp alkaline phosphotase (SAP)
  • 1% agarose gel
  • T4 DNA ligase and 10× T4 ligase buffer
  • Lambda in vitro packaging mix (e.g., MaxPlax, Epicentre cat. no. MP5110)
  • Mycobacteriophage buffer (MP buffer; see recipe)
  • E. coli HB101 cells (see protocol 10)
  • LB broth with and without hygromycin (150 mg/liter)
  • LB plates with hygromycin (150 mg/liter)
  • Qiagen miniprep kit (Qiagen cat. no. 27106)
  • 10× BSA
  • M. smegmatis mc2155
  • Middlebrook 7H9 broth without Tween‐80
  • Middlebrook top agar (see recipe)
  • Middlebrook 7H10 plates without Tween‐80
  • M. tuberculosis
  • Mycobacteriophage wash medium (see recipe)
  • 37° and 65°C water baths
  • Agarose gel electrophoresis apparatus
  • Microcentrifuge
  • 15‐ml snap‐cap tubes (Falcon cat. no. 2059 or equivalent)
  • Oak Ridge centrifuge tubes (Nalgene cat. no. 3115 or equivalent)
  • 0.45‐ and 5‐µm syringe filters
  • Spectrophotometer
  • 37° to 39°C heating block
  • 2‐ml screw‐cap tubes
  • Additional reagents and equipment for electroporation (see protocol 11)

Support Protocol 2: Lambda Phage Transduction of E. coli HB101

  Materials
  • E. coli HB101 (ATCC #33694)
  • LB broth supplemented with 10 MgSO 4 and 0.2% maltose
  • 10 mM MgSO 4
  • 37°C incubator with shaker
  • Spectrophotometer
  • 50‐ml conical tubes

Support Protocol 3: Electroporation of M. smegmatis mc2155

  Materials
  • M. smegmatis mc2155 cells
  • Middlebrook 7H9 broth or LB broth with 0.05% Tween
  • 10% glycerol with 0.05% Tween, 4°C
  • Transforming DNA
  • Medium plates with antibiotic (e.g., 10‐20 µg/ml Kanamycin at; at 50 µg/ml Hygromycin B)
  • Spectrophotometer
  • 50‐ml conical tubes, prechilled
  • 0.2‐cm electroporation cuvettes (GenePulser, BioRad cat. no. 165‐2086)
  • GenePulser electroporator (BioRad cat. no. 1652076)
  • 15‐ml snap‐cap plastic tube (Falcon cat. no. 2059 or equivalent)
  • Tabletop centrifuge

Basic Protocol 6: Transposon Mutagenesis of Mycobacterium tuberculosis

  Materials
  • M. tuberculosis culture
  • Mycobacteriophage (MP) buffer (see recipe)
  • High‐titer phage lysate containing transposon
  • Middlebrook 7H9 broth
  • Middlebrook 7H10 plates with 50 mg/liter hygromycin
  • Spectrophotometer
  • 15‐ml conical tube
  • Centrifuge (Sorvall RT 600 tabletop centrifuge or equivalent)
  • 37°C incubator
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Figures

Videos

Literature Cited

Literature Cited
   Bardarov, S., Bardarov, S. Jr., Pavelka, M.S. Jr., Sambandamurthy, V., Larsen, M., Tufariello, J., Chan, J., Hatfull, G., and Jacobs, W.R. Jr. 2002. Specialized transduction: An efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007‐3017.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513‐1523.
   Butcher, P.D. 2004. Microarrays for Mycobacterium tuberculosis. Tuberculosis (Edinb) 84:131‐137.
   Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E. 3rd, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., and Barrell, B.G. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537‐544.
   Connell, N.D. 1994. Mycobacterium: Isolation, maintenance, transformation, and mutant selection. Methods Cell Biol. 45:107‐125.
   Dietrich, G., Schaible, U.E., Diehl, K.D., Mollenkopf, H., Wiek, S., Hess, J., Hagens, K., Kaufmann, S.H., and Knapp, B. 2000. Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions. FEMS Microbiol. Lett. 186:177‐180.
   Kriakov, J., Lee, S., and Jacobs, W.R. Jr. 2003. Identification of a regulated alkaline phosphatase, a cell surface‐associated lipoprotein, in Mycobacterium smegmatis. J. Bacteriol. 185:4983‐4991.
   Mahenthiralingam, E. 1998. Extraction of RNA from mycobacteria. Methods Mol. Biol. 101:65‐75.
   Rubin, E.J., Akerley, B.J., Novik, V.N., Lampe, D.J., Husson, R.N., and Mekalanos, J.J. 1999. In vivo transposition of mariner‐based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad. Sci. U.S.A. 96:1645‐1650.
   Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall, A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a biosafety level 3 laboratory. J. Clin. Microbiol. 39:769‐771.
   Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T., and Jacobs, W.R. Jr. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4:1911‐1919.
   Stewart, G.R., Wernisch, L., Stabler, R., Mangan, J.A., Hinds, J., Laing, K.G., Young, D.B., and Butcher, P.D. 2002. Dissection of the heat‐shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:3129‐3138.
   Wards, B.J. and Collins, D.M. 1996. Electroporation at elevated temperatures substantially improves transformation efficiency of slow‐growing mycobacteria. FEMS Microbiol. Lett. 145:101‐105.
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