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Recombineering: Genetic Engineering in Bacteria Using ... · PDF fileRecombineering: Genetic Engineering in UNIT 1.16 Bacteria Using Homologous Recombination Lynn C. Thomason,1 James

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SECTION VSPECIALIZED TECHNIQUES

UNIT 1.16Recombineering: Genetic Engineering inBacteria Using HomologousRecombination

Lynn C. Thomason,1 James A. Sawitzke,2 Xintian Li,2 Nina Costantino,2

and Donald L. Court2

1Basic Science Program, GRCBL-Molecular Control & Genetics Section, Frederick NationalLaboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland2Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, NationalCancer Institute at Frederick, National Institutes of Health, Frederick, Maryland

ABSTRACT

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homol-ogous recombination using PCR products and synthetic oligonucleotides as substrates.This is possible because bacteriophage-encoded recombination proteins efficiently re-combine sequences with homologies as short as 35 to 50 bases. Recombineering allowsDNA sequences to be inserted or deleted without regard to location of restriction sites.This unit first describes preparation of electrocompetent cells expressing the recom-bineering functions and their transformation with dsDNA or ssDNA. It then presentssupport protocols that describe several two-step selection/counter-selection methods ofmaking 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) fromthe Escherichia coli chromosome or a co-electroporated DNA fragment. Additional pro-tocols describe methods to screen for unselected mutations, removal of the defectiveprophage from recombineering strains, and other useful techniques. Curr. Protoc. Mol.Biol. 106:1.16.1-1.16.39. C 2014 by John Wiley & Sons, Inc.

Keywords: recombineering bacteria homologous recombination bacteriophage Red system RecET Rac prophage selection/counter-selection

INTRODUCTION

Over the past decade, an in vivo technology has emerged that is precise, rapid, efficient,and more practical than standard in vitro recombinant genetic engineering techniques.This technology, termed recombineering, allows DNA sequences to be modified withoutregard to the presence or location of restriction sites, which are an essential componentof classical genetic engineering. The bacterial chromosome as well as bacterial episomescan be directly engineered in vivo by homologous recombination using linear DNAdonor substrates, such as PCR products or synthetic single-stranded oligonucleotides(ssDNA oligos). Gene knockouts, replacements, deletions, and point mutations can bemade; genes can be modified with tags, reporter fusions, and almost any imaginedconstruction. This in vivo genetic engineering is possible because bacteriophage-encodedrecombination functions efficiently recombine sequences with homologies as short as50 nucleotides (nt). The linear DNA donor substrate containing the desired changeis introduced by electroporation into bacterial strains that express the recombinationfunctions.

Current Protocols in Molecular Biology 1.16.1-1.16.39, April 2014Published online April 2014 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142727.mb0116s106Copyright C 2014 John Wiley & Sons, Inc.

Escherichia coli,Plasmids, andBacteriophages

1.16.1

Supplement 106

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Recombineering

1.16.2

Supplement 106 Current Protocols in Molecular Biology

The general flow chart in Figure 1.16.1 will help the researcher determine which recombi-neering pathway is most appropriate for the desired construct. The steps are summarizedbelow. First, define the goal of the recombineering project, which may be to insert a se-lectable marker, create point mutations or other small alterations, insert a non-selectablesegment of dsDNA, or retrieve dsDNA onto a plasmid vector. Depending on the goal,choose the appropriate recombineering system and generate the appropriate linear DNAsubstrate. Linear dsDNA substrates are generally made using PCR, while linear single-stranded substrates are synthetic oligos 70 nt in length with the desired alteration(s)centrally located. The linear DNA is introduced into electrocompetent bacteria (E. coli)pre-induced for the recombination functions.

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