Electrotransformation and Clonal Isolation of Rickettsia Species

Sean P. Riley1, Kevin R. Macaluso1, Juan J. Martinez1

1 Vector‐Borne Disease Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3A.6
DOI:  10.1002/9780471729259.mc03a06s39
Online Posting Date:  November, 2015
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Abstract

Genetic manipulation of obligate intracellular bacteria of the genus Rickettsia is currently undergoing a rapid period of change. The development of viable genetic tools, including replicative plasmids, transposons, homologous recombination, fluorescent protein‐encoding genes, and antibiotic selectable markers has provided the impetus for future research development. This unit is designed to coalesce the basic methods pertaining to creation of genetically modified Rickettsia. The unit describes a series of methods, from inserting exogenous DNA into Rickettsia to the final isolation of genetically modified bacterial clones. Researchers working towards genetic manipulation of Rickettsia or similar obligate intracellular bacteria will find these protocols to be a valuable reference. © 2015 by John Wiley & Sons, Inc.

Keywords: transformation; cell sorting; intracellular bacteria; clonal isolation; plaque assay; limiting dilution; immunofluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Electrotransformation of Rickettsia Species
  • Basic Protocol 2: Clonal Isolation of Rickettsia Species by Cell Sorting
  • Alternate Protocol 1: Clonal Isolation of Rickettsia Species by Limiting Dilution
  • Alternate Protocol 2: Clonal Isolation of Rickettsia Species by Plaque Assay
  • Basic Protocol 3: Confirmation of Rickettsia Infection by Immunofluorescence
  • Basic Protocol 4: Confirmation of DNA Transformation
  • Basic Protocol 5: Expansion and Cryopreservation of Rickettsia Clones
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Electrotransformation of Rickettsia Species

  Materials
  • 10 μl DNA (concentration: 0.5 to 2 mg/ml in H 2O)
  • 3 × 108 to 3 × 1010Rickettsia sp.
  • Confluent Vero cells
  • Hank's balanced salt solution plus glutamic acid and gelatin (HBSS++; see recipe)
  • 250 mM sucrose
  • Ice
  • Trypsin‐EDTA solution (Corning or see recipe)
  • Complete Dulbecco's modified Eagle's medium (cDMEM; see recipe)
  • cDMEM with 200 ng/ml rifampicin (diluted from 1000× stock solution, see recipe)
  • Diff‐Quik Stain Set (Siemens) or Giménez stain (Ammerman, et al. 2008, unit 3.5)
  • 175‐cm2 tissue culture flasks
  • 0.1‐cm‐gap electroporation cuvette
  • Electroporator
  • Tissue culture incubator
  • Rotator or shaker
  • Swinging‐bucket centrifuge
  • 50‐ml polypropylene tube

Basic Protocol 2: Clonal Isolation of Rickettsia Species by Cell Sorting

  Materials
  • Transformed Rickettsia from protocol 1
  • Vero cells
  • Complete Dulbecco's modified Eagle's medium (cDMEM; see recipe)
  • cDMEM with 200 ng/ml rifampicin (diluted from 1000× stock solution, see recipe)
  • 70% ethanol
  • Fluorophore‐conjugated anti‐Rickettsia antibody (RcPFA‐PerCP/Cy5.5, Lightning‐Link PerCP/Cy5.5 Conjugation Kit, Innova Biosciences)
  • Trypsin‐EDTA solution (Corning or see recipe)
  • Sucrose/phosphate/glutamate buffer (SPG; see recipe)
  • 96‐well tissue culture plates
  • 75‐cm2 flasks
  • 5‐ml syringes (four)
  • 15‐ , 19‐ , and 25‐G needles
  • 2‐μm syringe filter
  • Polypropylene tubes
  • Cell sorter
  • Tissue culture incubator
  • Swinging‐bucket centrifuge
  • Additional reagents and equipment for cytometry and cell sorting (Harkins, ; Harkins and Harrigan, ; Arnold and Lannigan, )

Alternate Protocol 1: Clonal Isolation of Rickettsia Species by Limiting Dilution

  Additional Materials (also see protocol 2)
  • Transformed Rickettsia from protocol 1
  • Vero cells

Alternate Protocol 2: Clonal Isolation of Rickettsia Species by Plaque Assay

  Additional Materials (also see protocol 2)
  • Transformed Rickettsia from protocol 1
  • Vero cells
  • cDMEM supplemented with 400 ng/ml (2×) rifampicin (diluted from 1000× stock solution, see recipe)
  • 1% agarose in water, autoclaved and maintained at 50°C
  • cDMEM with Neutral Red solution (Ammerman, et al. 2008, unit 3.5)
  • 6‐well plates (six)
  • Pasteur pipet (sterile)
  • Water bath (37°C and 50°C)
  • Inverted light microscope

Basic Protocol 3: Confirmation of Rickettsia Infection by Immunofluorescence

  Materials
  • Infected Vero cells from protocol 2 (three 96‐well plates)
  • 4% paraformaldehyde solution (see recipe)
  • Permeabilization buffer (PBS, 2% BSA, 0.1% Triton X‐100; see recipe)
  • Primary antibody solution (1:1000 RcPFA/2% BSA in PBS)
  • Secondary antibody solution (1:1000 donkey anti‐rabbit‐Alexafluor 488 [Life Technologies]/2% BSA in PBS)
  • Multichannel pipettor
  • Confocal microscope

Basic Protocol 4: Confirmation of DNA Transformation

  Materials
  • Rickettsia‐containing Vero cells from protocol 5
  • Trypsin‐EDTA solution (Corning or see recipe)
  • DNA isolation kit (PureLink Genomic DNA Mini Kit, Life Technologies)
  • Standard PCR buffers and polymerase
  • gfp uv‐specific DNA primers
  • DNA loading dye
  • 1% agarose DNA gel
  • Ethidium bromide
  • Tissue culture incubator
  • Thermal cycler
  • Electrophoresis apparatus
  • UV light box

Basic Protocol 5: Expansion and Cryopreservation of Rickettsia Clones

  Materials
  • Rickettsia from protocol 6
  • Sucrose/phosphate/glutamate buffer (SPG; filtered and sterilized, see recipe)
  • cDMEM supplemented with 200 ng/ml rifampicin (diluted from 1000× stock solution, see recipe)
  • Trypsin‐EDTA solution (Corning or see recipe)
  • 175‐cm2 flasks
  • Cryovials
  • −80°C freezer
  • Additional reagents and equipment for purification (unit 3.5, Ammerman et al., )
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Figures

Videos

Literature Cited

Literature Cited
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