RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida.
The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.
Lee
M D
MD
College of Veterinary Medicine, University of Georgia, Athens 30601, USA. lee.m@calc.vet.uga.edu
Henk
A D
AD
D000668:Ampicillin Resistance / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D008826:Microbial Sensitivity Tests; D016979:Pasteurella multocida / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000096:biosynthesis
D000900:Anti-Bacterial Agents; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase