Fig. 4

Deletion of the counterselection marker upp and the PHA polymerase subunit gene phaC. a Vector pUCTV2_Δupp for the deletion of upp, containing flanking genomic regions upstream and downstream of the gene (upp A′ and upp B′). The temperature-sensitive origin of replication (ori ^ts) prevents replication of the plasmid above temperatures of 42°C (tetR, tetracycline resistance gene; ampR, ampicillin resistance gene). b Vector pUCTV2_Upp_ΔphaC for the deletion of phaC, containing flanking genomic regions upstream and downstream of the gene (phaC A′ and phaC B′). It additionally contains the gene for the uracil phosphoribosyltransferase (upp) under the control of its natural promoter (P _upp ) which serves as a counterselection marker. c Agarose gel showing the PCR products using primers 9 and 10, flanking the upp gene and its promoter. Amplification with these primers results in DNA fragments of 865 bp size, if the upp-ORF is intact. The following templates were used: genomic DNA of MS941 (lane 1), pUCTV2_ Δupp (lane 2) and genomic DNA of upp deletion strain GHH1 (lane 3). The resulting fragment contained the upp gene truncated by 600 bp (lane 3) compared to the full-length gene (lane 1) (lane M: smartladder DNA marker (Eurogentec). d Agarose gel showing the PCR products using flanking primers 11 and 14. These bind approximately 1,000 bp upstream and downstram of the target gene. Amplification with these primers results in DNA fragments of ca. 3,000 bp size, if the phaC-ORF is intact. The following templates were used: genomic DNA of strain GHH1 (lane 1), pUCTV2_Upp_ΔphaC (lane 2) and genomic DNA of strain GHH3 (lane 3). The resulting fragment contained the phaC gene truncated by 1,088 bp (lane 3) compared to the full-length fragment (lane 1) (lane M: smartladder DNA marker). Additional bands in lane 2 stem from the plasmid used as DNA template.