Construction of New Campylobacter Cloning Vectors and a New Mutational Cat Cassette
NAVAL MEDICAL RESEARCH INST BETHESDA MD
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We have developed new Campylobacter shuttle vectors which are 6.5-6. 8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site MCS, the lacZalpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene KmR or CmR from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a KmR marker and 17 unique cloning sites in two different orientations in lacZalpha, allowing easy bluewhite color selection. Plasmids pRY111 and pRY112 contain a CmR gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CmR gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.
- Medicine and Medical Research