Genetic Transformation of Borrelia burgdorferi

Jenny A. Hyde1, Eric H. Weening2, Jon T. Skare1

1 Texas A&M University Health Science Center, College Station, Texas, 2 University of North Carolina, Chapel Hill, School of Medicine, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12C.4
DOI:  10.1002/9780471729259.mc12c04s20
Online Posting Date:  February, 2011
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Abstract

The development of robust genetic tools to manipulate Borrelia burgdorferi, the etiologic agent of Lyme disease, now allows investigators to assess the role(s) of individual genes in the context of experimental Lyme borreliosis. This unit is devoted to the description of experimental approaches that are available for the molecular genetic analysis of B. burgdorferi with an emphasis on cultivation, electrotransformation, selection of desired mutants, and genetic complementation of acquired mutants. The intent is to provide a consensus protocol that encapsulates the methodologies currently employed by the B. burgdorferi research community and describe pertinent issues that must be accounted for when working with these pathogenic spirochetal bacteria. Curr. Protoc. Microbiol. 20:12C.4.1‐12C.4.17. © 2011 by John Wiley & Sons, Inc.

Keywords: Spirochetes; Lyme borreliosis; mutation; allelic exchange; genetic complementation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transformation of B. burgdorferi
  • Basic Protocol 2: Antibiotic Selection by Semisolid Plating of B. burgdorferi Transformants
  • Alternate Protocol 1: Antibiotic Selection by Liquid Plating of B. burgdorferi Transformants
  • Basic Protocol 3: Recombinase‐Based Complementation of B. burgdorferi
  • Support Protocol 1: DNA Methylation of Transformed DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transformation of B. burgdorferi

  Materials
  • B. burgdorferi culture (see Table 12.4.1 for strains; see unit 12.1 and the Commentary of this unit for culture methods)
  • Complete 1× BSK medium (unit 12.1)
  • Phosphate‐buffered saline containing potassium (KPBS; see recipe), ice cold
  • HEPES/sucrose buffer: 8 mM HEPES, pH 7.4, with 272 mM sucrose, ice cold
  • 5 µg/µl DNA
    Table 2.0.1   MaterialsCommonly Used B. burgdorferisensu stricto Strains

    Strain Description and reference
    Wild‐type strains
    B31 Infectious low‐passage wild‐type strain isolated from Ixodes dammini and cloned by limiting dilution (Barbour, ; Burgdorfer et al., )
    297 Infectious low‐passage wild‐type strain isolated from human spinal fluid (Steere et al., )
    cN40 Infectious clonal low‐passage wild‐type strain isolated from adult I. dammini (Barthold et al., , )
    CA‐2‐87 I. pacificus isolate from Northern California (Schwan et al., )
    JD‐1 Isolated from Ixodes ticks (Piesman et al., )
    B31 derivatives
    B31 MI Derived from tick‐isolated B31 and passed through mouse (Casjens et al., )
    B31‐A3 Clonal B31 infectious and low‐passage; plasmids and infectivity maintained with transformation of shuttle or suicide vectors (Elias et al., )
    MSK5 Clonal infectious low‐passage strain isolated from C3H/HeN mouse skin (Labandeira‐Rey and Skare, )
    MSK7 Clonal infectious low‐passage strain isolated from C3H/HeN mouse skin, lacking lp28‐4 (Labandeira‐Rey and Skare, )
    MSK10 Clonal low‐passage strain unable to establish persistent infection; lacking lp28‐1, isolated from C3H/HeN mouse skin (Labandeira‐Rey and Skare, )
    ML23 Clonal non‐infectious low‐passage strain lacking lp25, isolated from nonclonal P48 B31 culture (Labandeira‐Rey and Skare, )
    5A# Clonal isolates with variable plasmid composition and infectivity (Purser and Norris, )
    5A4 Clonal infectious isolate containing all plasmids (Purser and Norris, )
    5A10 & 5A14 Clonal infectious isolate lacking lp25 and lp56 (Purser and Norris, )
    5A18 Clonal infectious isolate lacking lp28‐4 and lp56 (Purser and Norris, )
    5A4 NP1 Clonal infectious isolate containing all plasmids (5A4) and bbe02 disrupted with P flgBkanR (Kawabata et al., )
    5A18 NP1 5A18 with bbe02 disrupted by a P flgBkanR cassette (Kawabata et al., )
    297 derivatives
    BbAH130 Clonal infectious low‐passage strain isolated from plating polyclonal 297 (Yang et al., )
    PL133 Clonal infectious low‐passage strain isolated from ear punch biopsies of mice infected with polyclonal 297 (Revel et al., )
    BbDTR630 Derived from PL133 with a P flgBkanR cassette cloned into lp25 (Blevins et al., )
    297‐LK Non‐infectious clonal isolate derived from AH130 and lacking lp28‐1; lacI and P flgBkan cloned into bbe02 on lp25 (Gilbert et al., )
    c155 Infectious low‐passage strain isolated from nonclonal 297 by two rounds of plating and colony extraction (Eggers et al., )
    CE162 Clonal infectious low‐passage strain derived from wild‐type 297 (Caimano et al., )
    cN40 derivative
    D10/E9 Infectious low‐passage clonal isolate (Parveen et al., )

  • Sterile centrifuge bottles, 50‐ml conical tubes, pipets, and pipet tips (aerosol‐resistant)
  • Electroporator (e.g., Bio‐Rad Gene Pulser) with 0.2‐cm cuvettes

Basic Protocol 2: Antibiotic Selection by Semisolid Plating of B. burgdorferi Transformants

  Materials
  • 1.7% (w/v) molecular‐grade agarose
  • 2× BSK‐II medium (unit 12.1), prewarmed to 48°C
  • Normal rabbit serum (NRS)
  • Antibiotics, filter sterilized
  • Transformed B. burgdorferi culture (see protocol 1)
  • Autoclave
  • 48°C water bath
  • Disposable sterile Petri dishes, 100 mm o.d. × 15 mm
  • Disposable sterile pipets, pipet tips (aerosol resistant), and 15‐ml conical tubes

Alternate Protocol 1: Antibiotic Selection by Liquid Plating of B. burgdorferi Transformants

  Materials
  • Complete 1× BSK (unit 12.1)
  • 0.5% (w/v) phenol red, filter sterilized
  • Antibiotics, filter sterilized
  • Transformed B. burgdorferi culture (see protocol 1)
  • Multichannel pipet
  • Sterile 96‐well plate, basin, and pipet tips (aerosol resistant)
  • Plate tape (e.g., Petri Seal)

Basic Protocol 3: Recombinase‐Based Complementation of B. burgdorferi

  Materials
  • Borrelial destination shuttle vector (Table 12.4.2)
  • ccdB survival T1R E. coli competent cells (Invitrogen)
  • PCR fragment of gene of interest
  • Entry vector pCR8 or one of many pENTR vectors (Invitrogen)
  • LR Clonase II (Invitrogen)
  • 2 µg/µl proteinase K
  • Competent DH5α E. coli cells
    Table 2.0.2   MaterialsPlasmids for Genetic Manipulation of B. burgdorferi

    Plasmid Selection a Description
    Shuttle vectors
    pBSV2 kanR Shuttle vector containing three ORF's from cp9 required for plasmid replication and distribution (Stewart et al., )
    pBBE22 kanR pBSV2 containing bbe22/pncA fragment to restore infectivity to strains lacking lp25 (Purser et al., )
    pBBE22gate kanR Modified pBBE22 containing Gateway destination vector attR1 and attR2 sites from Invitrogen's pDEST17 (Weening et al., )
    pKFSS1 strepR Streptomycin‐resistant borrelial shuttle vector derived from pBSV2 (Frank et al., )
    pKFSS1gate strepR Modified pKFSS1 containing Gateway destination vector attR sites cloned into the SphI site within the MCS (Weening and Skare, unpublished)
    pCADDY strepR Shuttle vector derived from pBBE22, replacing kanamycin resistance with streptomycin resistant (Hyde et al., )
    pCADDYgate strepR Derived from pBBE22gate (Weening et al., ), replacing kanamycin resistance with streptomycin resistance (Hyde and Skare, unpublished)
    pJD1 strepR pKFSS1 derivative with reduced P flgBaadA region (Blevins et al., )
    pJD7 strepR pJD1 derivative with terminator inserted and P lac removed (Blevins et al., )
    pJD44 kanR pJD7 derived plasmid replaced specR/strepR with kanR (Blevins et al., )
    pBSV2G gentR Gentamicin‐resistant shuttle vector derived from pBSV2 (Elias et al., )
    pBSV2Ggate gentR Modified pBSV2G containing Gateway destination vector attR sites at SphI site in MCS (Weening and Skare, unpublished)
    pBSV2SynG courR Shuttle vector derived from pBSV2 encoding a synthetic gyrB (Elias et al., )
    pCE310 kanR Shuttle vector with cp32 loci required for replication and distribution (Eggers et al., )
    pCB52 kanR Contains replicon and telomere ends from lp17; replicates as circular plasmids in E. coli and as a linear plasmid in B. burgdorferi (Beaurepaire and Chaconas, )
    pBSV17 kanR Shuttle vector derived from pBSV2 with gene required for replication from lp17 (Beaurepaire and Chaconas, )
    pGK12 ermR Broad‐host plasmid able to propagate in Gram‐positive and Gram‐negative bacteria including B. burgdorferi (Sartakova et al., )
    Allelic exchange vectors
    pJH333 gentR A multicloning site and P flgBgentR cassette flanked by DNA regions for allelic exchange into the borrelial chromosome between bb0445 and bb0446; based on the construct reported by Li et al., (Hyde and Skare, unpublished)
    pMC1667 none b Suicide vector with multicloning site flanked by cp26 regions for allelic exchange (Dunham‐Ems et al., )
    pMC1916 gentR Suicide vector derived from pMC1667 with P flgBgentR and P flaBgfp in divergent orientation are flanked by cp26 regions for allelic exchange (Dunham‐Ems et al., )
    pSPC‐G gentR Suicide vector with P flgBgentR and P flaBgfp in divergent orientations are flanked by cp26 regions for allelic exchange (Kenedy et al., )

     aSelection is in reference to antibiotic resistance in Borrelia burgdorferi only; antibiotic resistance for usage in E. coli is not indicated. Cour, coumermycin; erm, erythromycin; gent, gentamicin; kan, kanamycin; strep, streptomycin.
     bNo selectable marker is incorporated for usage in B. burgdorferi; plasmid does confer resistance to kanamycin and ampicillin for selection in E. coli.

Support Protocol 1: DNA Methylation of Transformed DNA

  Materials
  • CpG methyltransferase (M.SssI; New England Biolabs)
  • NEBuffer 2 (New England Biolabs)
  • S‐Adenosylmethionine (SAM)
  • DNA to be used for electroporation
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
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Figures

Videos

Literature Cited

Literature Cited
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