edoc

A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica

Kaniga, K. and Delor, I. and Cornelis, G. R.. (1991) A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene, Vol. 109, H. 1. S. 137-141.

Full text not available from this repository.

Official URL: http://edoc.unibas.ch/dok/A5259243

Downloads: Statistics Overview

Abstract

A new suicide vector (pKNG101) that facilitates the positive selection of double recombination events in Gram-bacteria has been developed. It contains a conditional origin of replication (oriR6K), the strAB genes encoding the streptomycin phosphotransferase (SmR), an origin of transfer (mobRK2), the sacB gene mediating sucrose sensitivity, and multiple cloning sites. It was used to mutate the blaA gene of Yersinia enterocolitica, by marker-exchange mutagenesis. To do this, we have first cloned into the suicide vector pKNG101, a 2.5-kb fragment of Y. enterocolitica chromosomal DNA encoding the 20-kDa beta-lactamase A. Gene blaA was then mutated in vitro by insertion of luxAB, which resulted in pKNG105. The disrupted blaA gene was then reintroduced into Y. enterocolitica chromosome by homologous recombinations in two steps. First, E. coli SM10 lambda pir (pKNG105) was mated with strains of Y. enterocolitica. This led to the integration of pKNG105 into the chromosome, by a single homologous recombination event. The transconjugants, selected for SmR, were sensitive to sucrose due to the synthesis of levans (toxic compounds), catalysed by levansucrase, the product of sacB. For the second step, a single colony from the first step was grown in rich medium deprived of antibiotic, allowing the occurrence of a second crossing-over that replaced the wild-type allele blaA with the mutant one, and then excised the plasmid-borne sacB from the chromosome. Such blaA mutants were selected on their ability to grow on TSA medium containing 5% sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Molecular Microbiology (Cornelis)
UniBasel Contributors:Cornelis, Guy R.
Item Type:Article, refereed
Bibsysno:Link to catalogue
Publisher:Elsevier/North-Holland
ISSN:0378-1119
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:20
Deposited On:22 Mar 2012 13:22

Repository Staff Only: item control page