Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation
Fig 2
Scheme for the construction of the unmarked SC1401 ΔhtrA strain.
The plasmid pMDHKS was generated by inserting the sacB fragment (amplified from the plasmid pEMOC2 using primer pairs P7/P8) into the plasmid pMDHK by inverse PCR (using primer P9 and P10) and In-Fusion cloning. The plasmid pMDH was obtained by inverse PCR (using primer P11 and P12) and In-Fusion cloning. To construct the unmarked SC1401 ΔhtrA strain, the kan-sacB cassette was integrated into the SC1401 chromosome and the htrA gene was replaced by the first allelic replacement. Next, the linearized plasmid pMDH was employed in the second allelic replacement to remove the kan-sacB cassette from the SC1401ΔhtrA::kan-sacB chromosome, leaving an unmarked htrA deletion strain.