Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system

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Author: Emmerson, JR
Author: Roe, AJ
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Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system

James R. Emmerson¹, David L. Gally¹ and Andrew J. Roe¹^*

¹ Centre for Infectious Diseases, University of Edinburgh. The Chancellors Building, 49 Little France Crescent, EH16 4SB. United Kingdom.

* To whom correspondence should be addressed: Andrew J. Roe, Centre for Infectious Diseases, University of Edinburgh. The Chancellors Building, 49 Little France Crescent, EH16 4SB. United Kingdom. Email: aroe@vet.ed.ac.uk

Biol. Proced. Online 2006;8:153-162. doi:10.1251/bpo123
Submitted: July 05, 2006; Accepted: September 12, 2006; Published: September 27, 2006.

Indexing terms: Gene Deletion; Escherichia coli O157:H7.

Figure 1 Enlarged

Fig. 1:

(Adapted from Blomfield et al. 1991). The transfer of the sacB-kan cassette into the chromosome requires two recombination events leading to plasmid integration followed by plasmid excision. Step 1: plasmid integrates into the wild-type strain at the non permissive temperature for plasmid replication (42°C). Steps 2 and 3: plasmid integrates are grown at 28°C in the presence of kanamycin to enrich for bacteria that have excised and cured the plasmid, leaving the cassette on the chromosome. Step 4: Growth on media containing LBC or LBK. Bacteria that can only grow on LBK are successful constructs of the intermediate strain.

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