earticle

영어

Cyanobacteria are photosynthetic bacteria whose characteristics are determined by fixing CO2, using photon energy and evolving O2 gas from water splitting. Owing to these ‘environment- friendly’ features, they have potentials to be exploited for clean, renewable fuel production, especially biohydrogen. To improve cyanobacterial strain for efficient hydrogen production, genetic manipulation technique is needed. Thus, in the present work, we constructed a shuttle expression vector for Synechocystis PCC 6803 (Syn6803) as a first footstep. It consists of several parts which came from different sources. The replication origin (oriV), origin of transfer (oriT), and associated genes (repA,B,C) were cloned as a 5.6kbp fragment from RSF-1010, a broad host range plasmid, allowing to replicate autonomously in some cyanobacterial strains including Syn6803. The commercial vectors, pET-28b (Novagen) and pTrcHisC (Invitrogen), provided kanamycin resistance gene, multiple cloning sites (MCS), and other accessory sequences. The nickel-inducible strong promoter was PCRcloned from nickel response operon (nrs) within the genome of Syn6803. The nickel metal for induction is also essential component for the activity of NiFehydrogenase which is the enzyme responsible for hydrogen production. We introduced green fluorescent protein (GFP) gene into the MCS region of the complete vector which was named pRKNrs. We will present functional expression of reporter GFP in Syn6803 strains to confirm successful construction.