Cloning of Polyhydroxybutyrate operon and Phage Фx174 E Gene in Separate Plasmids and Analysis of E Gene Function in Facilitating Polyhydroxybutyrate Production in E.coli

Polyhydroxybutyrate is a biodegradable plastic that has widespread application in various industries and in the field of medical equipment production. In E.coli, the most cost of Polyhydroxybutyrate production (PHB) is related to its extraction and purification processes. E gene of Phage Фx174 with conformation of membrane tunnel is used for lysing the hosted bacteria. This study was conducted to investigate the function of E gene in conformation of membrane tunnel and release of produced PHB in E.coli. In this study, after amplification with polymerase chain reaction (PCR), PHB operon was cloned by pUC18 while its expression controlled by operon promoter. E gene was amplified with PCR and cloned by pMR103 plasmid while it’s expression controlled by T7 promoter. Lysis process and conformation of membrane tunnel were evaluated by electronic microscope. Analysis of produced PHB was performed by gas chromatography and Sudan staining. With activation of E gene and lysis of bacteria, optical density of specimen decreased. In the analysis of bacterial debris which achieved from E.coli medium by electronic microscope, we observed membrane pores in lysed bacteria. The results of gas chromatography and Sudan staining showed that PHB granules have been released to medium by conformation of membrane tunnel. Lysis E gene can be used for decreasing the cost and facilitating the extraction of PHB in E.coli that can be applied for manufacture of disposable medical and operating room equipment.