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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Curr Protoc Microbiol. 2020 Sep;58(1):e111. doi: 10.1002/cpmc.111

Figure 1: Vector and procedure used for generation of a gene deletion cassette.

Figure 1:

(A) Presented is pK19mobsacB, a non-replicating vector in C. diphtheriae used to generate gene deletion mutants in this organism. This vector contains a kanamycin resistant gene (KmR), sacB, mutiple cloning sites (MCS) within a lacZα fragment, and origins of replicon oriV and oriT; adapted from (H. Ton-That & O. Schneewind, 2003). (B) To delete a gene of interest (red), two sets of primers (F1/R1 and F2/R2) are designed for two PCR reactions PCR1 and PCR2 that generate 1-kb fragments A and B, respectively. Restriction enzyme sites are incorporated into primers F1 and R2 (orange and pink). Primers R1 and F2 contain a complementary sequence permitting annealing of fragments A and B to produce fragment AB during the third PCR reaction with primers F1 and R2.