PHB synthase encoded by phbC
gene, is the last enzyme in PHB
biosynthesis pathway in Alcaligenes eutrophus. A. eutrophus PTCC 1615 is well known for its polyhydroxybutyrate production capability. In this study, a 2190 bp DNA fragment including phbC
gene, its promoter and terminator region were isolated from A. eutrophus PTCC 1615. Primers were designed with Oligo7 and Gene runner software, using genome sequence of A. eutrophus H16 available in nucleotide database of NCBI following which they were evaluated by NCBI Primer Blast. To obtain the complete sequence of the target fragment, internal primers were designed and evaluated using the same protocol. The existence of promoter and terminator regions in isolated DNA fragment was appropriate for over expression in A. eutrophus at the same time with its own phbC
gene, without using an inducer. PCR was set up with Pfu DNA polymerase. This DNA polymerase exhibits proofreading activity and its error rate is just 2.6×10-6 nucleotide per cycle of PCR. Hence, the fidelity would be ensured in the PCR products in the process of cloning.
The size and sequence of target DNA sequences were confirmed by agarose gel electrophoresis and the sequencing data, respectively. The isolated DNA segment exhibited 99% identity with A. eutrophus H16 phbC
gene in NCBI Nucleotide database.