Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Lynn C. Thomason1, James A. Sawitzke2, Xintian Li2, Nina Costantino2, Donald L. Court2

1 Basic Science Program, GRCBL‐Molecular Control & Genetics Section, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, Maryland, 2 Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.16
DOI:  10.1002/0471142727.mb0116s106
Online Posting Date:  April, 2014
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Abstract

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two‐step selection/counter‐selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co‐electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Curr. Protoc. Mol. Biol. 106:1.16.1‐1.16.39. © 2014 by John Wiley & Sons, Inc.

Keywords: recombineering; bacteria; homologous recombination; bacteriophage λ; λ Red system; RecET; Rac prophage; selection/counter‐selection

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Making Electrocompetent Cells and Transforming with Linear DNA
  • Support Protocol 1: Selection/Counter‐Selections for Gene Replacement
  • Support Protocol 2: Retrieval of Alleles onto a Plasmid by Gap Repair
  • Support Protocol 3: Screening for Oligo Recombinants by PCR
  • Support Protocol 4: Other Methods of Screening for Unselected Recombinants
  • Support Protocol 5: Modifying Multicopy Plasmids with Recombineering
  • Support Protocol 6: Screening for Unselected Plasmid Recombinants
  • Basic Protocol 2: Removal of the Prophage by Recombineering
  • Basic Protocol 3: Curing Recombineering Plasmids Containing a Temperature‐Sensitive Replication Function
  • Alternate Protocol 1: Recombineering with an Intact λ Prophage
  • Alternate Protocol 2: Targeting an Infecting Phage λ with the Defective Prophage Strains
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Making Electrocompetent Cells and Transforming with Linear DNA

  Materials
  • Purified PCR product with ∼50 bases of flanking homology or oligonuclotide primers with ∼35 bases of flanking homology on either side of desired change (also see unit )
  • Bacterial strain expressing the defective lambdoid prophage recombination system λ Red (Table 1.16.1; strains available from Court Laboratory Website: http://redrecombineering.ncifcrf.gov/)
  • LB medium and plates (unit ), without antibiotic
  • Medium lacking carbon source: M9 medium (unit ) or 1× TM buffer ( )
  • Selective plates (unit )—minimal plates if selecting for prototrophy or rich plates containing antibiotic (depending on drug cassette used):
    • 30 µg/ml ampicillin
    • 30 µg/ml kanamycin
    • 10 µg/ml chloramphenicol
    • 12.5 µg/ml tetracycline
    • 50‐100 µg/ml spectinomycin
  • 30° to 32°C incubator
  • 30° to 32°C shaking incubator or roller
  • 32° and 42°C shaking water baths
  • 125‐ and 250‐ml Erlenmeyer flasks, preferably baffled
  • Refrigerated, low‐speed centrifuge with Sorvall SA‐600 rotor (or equivalent)
  • 35‐ to 50‐ml plastic centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • 0.1‐cm electroporation cuvettes (Bio‐Rad), chilled
  • Electroporator (e.g., Bio‐Rad E. coli Pulser)
  • Micropipettor and 1000‐µl pipet tips
  • 18 × 150‐mm sterile borosilicate glass culture tubes
  • 15‐ml sterile culture tubes, optional
  • Additional reagents and equipment for PCR (unit ), agarose gel electrophoresis of DNA (units & ), purification of DNA by ethanol precipitation (unit ; optional; commercially available PCR cleanup kit may be substituted), electroporation (unit ), isolation of bacterial colonies by streaking (unit ), restriction enzyme digestion (unit ), and DNA sequencing (Chapter 7)

Support Protocol 1: Selection/Counter‐Selections for Gene Replacement

  Additional Materials(also see protocol 1)
  • Bacterial strain for specific selection/counter‐selection:
    • cat‐sacB: any recombineering strain can be used
    • tetA‐sacB: the Red functions should be expressed from a heat curable plasmid rather than from a prophage
    • galK: use strain SW102, available from Frederick National Laboratory for Cancer Research: http://ncifrederick.cancer.gov/research/brb/recombineeringInformation.aspx
  • Appropriate template for amplification of selectable/counter‐selectable cassette:
    • cat‐sacB: use bacterial strain TUC01 (DY329 with a cat‐sacB insertion on the E. coli chromosome), available from the Court Laboratory Website: http://redrecombineering.ncifcrf.gov/
    • tetA‐sacB: E. coli strain T‐SACK, available from the Court Laboratory
    • galK: the galK expression cassette has been cloned onto BAC pBeloBAC11, also available from Frederick National Laboratory for Cancer Research
  • Hybrid primers to amplify cassettes—all hybrid primers should have 50 bases of 5′ homology for targeting to the desired location:
    • cat‐sacB
    • Primer L sacB: 5′‐homology sequence‐ATC AAA GGG AAA ACT GTC CAT AT‐3′
    • Primer R cat: 5′‐homology sequence‐TGT GAC GGA AGA TCA CTT CG‐3′
    • tetA‐sacB
    • Primer L sacB: same as for cat‐sacB
    • Primer R tetA: 5′‐homology sequence‐ TCC TAA TTT TTG TTG ACA CTC TA‐3′
    • galK
    • GalK forward primer: 5′‐homology sequence‐CCT GTT GAC AAT TAA TCA TCG GCA‐3′
    • GalK reverse primer: 5′‐homology sequence‐TCA GCA CTG TCC TGC TCC TT‐3′
  • Invitrogen Platinum High Fidelity enzyme
  • Selective media for inserting and removing cassettes:
    • cat‐sacB selection, use LB‐Cm plates: LB plates (unit ) containing 10 µg/ml chloramphenicol; for counter‐selection, use LB plates (unit ) lacking NaCl but containing 6% (w/v) sucrose
    • tetA‐sacB selection, use LB‐Tet plates: LB plates (unit ) containing 12.5 µg/ml tetracycline; for counter‐selection, use Tet/SacB plates (see recipe)
    • galK selection, use M63 minimal D‐galactose plates containing biotin and leucine, with chloramphenicol for BAC selection (see recipe); for screening, use MacConkey galactose plates (see recipe); for counter‐selection, use M63 minimal glycerol with 2‐deoxygalactose (DOG), biotin and leucine, and chloramphenicol for BAC selection (see recipe)
  • Thermal cycler (MJ Research PTC‐100)

Support Protocol 2: Retrieval of Alleles onto a Plasmid by Gap Repair

  Additional Materials (also see protocol 1)
  • Synthetic chimeric primers providing homology to sequence flanking gene of choice and to the plasmid sequence to be amplified
  • Plasmid onto which sequence of choice is to be rescued
  • Restriction enzyme(s) (unit ) that do not cut within plasmid region to be amplified
  • Bacterial strain SIMD63 or other RecET expression system (with full length recE) for recombining two PCR products

Support Protocol 3: Screening for Oligo Recombinants by PCR

  Additional Materials (also see protocol 1)
  • 70‐mer oligo containing desired mutation

Support Protocol 4: Other Methods of Screening for Unselected Recombinants

  Additional Materials (also see protocol 1)
  • Flanking primers for PCR analysis of mutation of interest (also see unit )
  • Additional reagents and equipment for colony hybridization (e.g., unit )

Support Protocol 5: Modifying Multicopy Plasmids with Recombineering

  Additional Materials (also see protocol 1)
  • Plasmid to be modified: monomer species, freshly isolated from recA mutant host such as DH5α; determine plasmid DNA concentration by A 260
  • recA mutant bacterial strain expressing Red functions (e.g., DY331 or DY380; see Table 1.16.1)
  • Appropriate selective plates, if needed (when selecting for drug resistance, use drug concentrations appropriate for the multicopy plasmid used)
  • Additional reagents and equipment for plasmid miniprep (unit ) and agarose gel electrophoresis (unit )

Support Protocol 6: Screening for Unselected Plasmid Recombinants

  Additional Materials (also see protocol 1)
  • Cells expressing Red but mutant for host mismatch repair system: e.g., HME63, HME64, and/or HME68 (see Table 1.16.1) or cells expressing Red and competent for host mismatch repair and an oligonucleotide that creates mismatches refractory to mismatch repair when annealed to target DNA strand
  • High‐efficiency cloning strain (lacking the Red system)

Basic Protocol 2: Removal of the Prophage by Recombineering

  Materials
  • Oligonucleotide primers for amplifying the bacterial attB site:
    • 5′ GAG GTA CCA GGC GCG GTT TGA TC 3′
    • 5′ GTT GCC GAT GTG CGC GTA CTG 3′
  • E. coli K12 strain lacking the prophage (e.g., W3110), but containing the attB and biotin (bio) genes
  • M63 minimal glucose plates (see recipe) with and without biotin
  • Additional reagents and equipment for recombineering (see protocol 1)

Basic Protocol 3: Curing Recombineering Plasmids Containing a Temperature‐Sensitive Replication Function

  Materials
  • Overnight culture of confirmed recombinant strain
  • Rich broth
  • 50‐ or 125‐ml baffled Erlenmeyer flask
  • 37° to 42°C shaking water bath
  • Additional reagents and equipment for recombineering (see protocol 1)

Alternate Protocol 1: Recombineering with an Intact λ Prophage

  Additional Materials (also see protocol 1)
  • Bacterial lysogen carrying the λ cI857 bacteriophage of choice as a prophage
  • Chloroform
  • 1× TM buffer ( )
  • 82‐mm nitrocellulose filters, sterile
  • Sterile forceps
  • 39°C shaking water bath
  • Additional reagents and equipment for plating λ phage to generate plaques (plaque purification; unit ), PCR (unit ), and DNA sequencing (Chapter 7)

Alternate Protocol 2: Targeting an Infecting Phage λ with the Defective Prophage Strains

  Additional Materials (also see protocol 1)
  • Host strain with defective prophage
  • 10% (w/v) maltose stock solution, filter sterilized
  • 1× TM buffer ( )
  • High‐titer lysate of bacteriophage to be engineered
  • PCR product with desired sequence changes and flanking homology to the target on the phage chromosome
  • Chloroform
  • Lambda plates and lambda top agar (see unit , adjust NaCl to 5 g per liter in both plates and top agar)
  • Appropriate bacterial indicator strain
  • Additional reagents and equipment for working with λ bacteriophages (units )
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Literature Cited

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