Abstract
A plasmid vector was designed, constructed, and used for the repeated retrieval of the bvgA gene from a number of Bordetella pertussis strains that, due to mutations in this gene, exhibited interesting phenotypes regarding the regulation of virulence genes. The vector was used in a scheme called allelic retrieval that exploits two cross-overs between cloned plasmid and native chromosomal sequences flanking the bvgA gene. This scheme is very similar to allelic exchange through the use of plasmid suicide vectors, but in the case presented here, the non-replicating plasmid that has received the chromosomal gene is recovered, rather than being allowed to be lost due to segregation. Incorporation of the counterselectable sacB gene of Bacillus subtilis in place of the plasmid copy of bvgA allows selection, after recovery in Escherichia coli, for only those plasmids that have retrieved the chromosomal bvgA gene. The validity of this approach was demonstrated by the retrieval of bvgA alleles with distinctive physical markers, as well as by the reintroduction of retrieved bvgA alleles to demonstrate that they conferred the expected phenotypes. It is expected that this approach will be applicable to the analysis of other genes in other b...Continue Reading
References
Nov 24, 1979·Nucleic Acids Research·H C Birnboim, J Doly
Mar 1, 1992·Molecular Microbiology·S KamounC I Kado
May 1, 1992·Molecular Microbiology·H P Schweizer
Jun 1, 1991·Molecular Microbiology·I C BlomfieldB I Eisenstein
Dec 20, 1991·Gene·K KanigaG R Cornelis
Jan 1, 1986·Gene·S StibitzS Falkow
Jan 1, 1987·Gene·J L Ried, A Collmer
Jan 1, 1981·Nature·G B Ruvkun, F M Ausubel
Jun 1, 1995·Molecular Microbiology·P SanderE C Böttger
Oct 1, 1993·Molecular Microbiology·H L MobleyJ W Warren
Dec 22, 1993·Gene·S Geissler, M Drummond
Oct 1, 1993·Vaccine·J R ButtertonS B Calderwood
May 15, 1993·Gene·J Quandt, M F Hynes
Feb 22, 1996·Gene·K Skorupski, R K Taylor
Jun 1, 1997·Microbiology·Rongdian Fud, Gerrit Voordouw
Citations
Jan 31, 2012·Protein Engineering, Design & Selection : PEDS·Cathie M OverstreetGregory A Weiss
May 9, 1998·Journal of Bacteriology·S Stibitz
May 14, 2020·PLoS Pathogens·Qing ChenScott Stibitz