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Nucleic Acids Res. 2014 Mar;42(5):e37. doi: 10.1093/nar/gkt1339. Epub 2013 Dec 24.

Improved seamless mutagenesis by recombineering using ccdB for counterselection.

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Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China, Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany, Shandong University-Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China, Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany and Gene Bridges GmbH, Building C2.3, Saarland University, 66123 Saarbrücken, Germany.


Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.

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