Fig. 1
From: Multivalent poultry vaccine development using Protein Glycan Coupling Technology
Generation of a suicide plasmid for chromosomal integration of the C. jejuni pgl locus in APEC χ7122 via homologous recombination a Schematic representation of the cloning strategy used to construct the pSECpgl suicide vector used for integration of the C. jejuni pgl locus between genes gidB and atpI in APEC genome. A Gibson assembly reaction with seven fragments was assembled. IntUP (~ 1 kb left homology arm for integration), C. jejuni pgl operon consisting of 3 fragments of ~ 5 kb each (pgl1-3), kanamycin selection marker surrounded by FRT sites, and IntDN (~ 1 kb right homology arm for integration) were PCR amplified and assembled into linearised pCVD442 plasmid between base coordinates 3633 and 4253 replacing the IS1 element. b Junction PCRs to assess correct assembly of pSECpgl. Lanes 1 to 8—diagnostic junction PCR products from eight AmpR, KanR Gibson assembly transformants. The expected PCR product size along with primer pairs used are shown. Clones 1 and 7 showed expected PCR products for all five junction PCRs tested. c)Diagnostic restriction analysis of pSECpgl plasmids isolated from colonies 1, 6 and 7 shown in b